Selective Poly-N-Substituted Glycine Antibiotics

ABSTRACT

Antimicrobial peptoid compounds and related compositions as can be used against bacteria effectively and selectively.

This application is a continuation of and claims priority benefit fromapplication Ser. No. 12/378,034 filed Feb. 9, 2009 and issued as U.S.Pat. No. 8,445,632 on May 21, 2013, which claimed priority benefit fromapplication Ser. No. 61/065,189 filed Feb. 8, 2008—each of which isincorporated herein by reference in its entirety.

This invention was made with government support under Grant Nos. 1R01HL67984-01 and AI007266 awarded by the National Institutes of Health andContract No. DE-AC02-05CH11231 awarded by the U.S. Department of Energy.The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Natural antimicrobial peptides (AMPs) defend a wide array of organismsagainst bacterial invaders and show potential as supplements for orreplacements of conventional antibiotics, since few bacteria haveevolved resistance to them. Many AMPs kill bacteria by permeabilizationof the cytoplasmic membrane, causing depolarization, leakage, and death,whereas others target additional anionic bacterial constituents (e.g.DNA, RNA, or cell wall components). Bacterial resistance to AMPs is rareprobably because they have evolved along with the resistance mechanismsdesigned to evade them; furthermore, the targets of many AMPs (e.g.bacterial plasma membranes, anionic intracellular macromolecules) aresufficiently general that changes to their sequence can be made thatsubvert resistance, yet have negligible impact on overall functionality.

Although AMPs have been actively studied for decades, they have yet tosee widespread clinical use. This is in part due to the vulnerability ofmany peptide therapeutics to rapid in vivo degradation, whichdramatically reduces their bioavailability. Non-natural mimics of AMPscan circumvent the proteolytic susceptibility of peptides whileretaining their beneficial features. The short (<40 amino acids), simplestructure of AMPs in the cationic, linear, α-helical class, whichincludes the well-known magainins, are especially amenable to mimicry.β-peptide mimics of these AMPs have been successfully created withantibacterial and non-hemolytic in vitro activity. Poly-N-substitutedglycines (peptoids) comprise another class of peptidomimetics, and areisomers of peptides in that peptoid side chains are attached to thebackbone amide nitrogen rather than to the α-carbon. More than any ofthe other peptidomimetic systems under study, including β-peptides,β-peptoids, oligoureas, and oligo(phenylene ethynylene)s, peptoids areparticularly well-suited for AMP mimicry because they are easilysynthesized on solid phase (using conventional peptide synthesisequipment) with access to diverse sequences at relatively low cost. Byway of an elegant submonomer synthetic method, any chemicalfunctionality available as a primary amine can be incorporated, whetherit be an analog of a proteinogenic amino acid or a totally non-naturalmoiety; thus, peptoids are highly and finely tunable. Furthermore, theyare protease-resistant, and can be designed to form amphipathic helicesthat resist thermal and chaotropic denaturation.

The poly-N-substituted glycine structure of peptoids precludes bothbackbone chirality and intrachain hydrogen bonding; nevertheless,peptoids can be driven to form stable helical secondary structures viaperiodic incorporation of bulky, α-chiral side chains. X-ray, NMR, andCD studies of peptoid oligomers have shown that incorporation ofhomochiral side chains can give rise to polyproline type-1-like heliceswith a periodicity of ˜3 monomers per turn and a helical pitch of6.0-6.7 Å. The three-fold periodicity of the peptoid helix facilitatesthe design of facially amphipathic structures similar to those formed bymany AMPs; for example, the trimer repeat (X—Y—Z)_(n) forms a peptoidhelix with three faces, composed of X, Y, and Z residues, respectively.

Amphipathic secondary structures in which residues are segregated intohydrophobic and cationic regions are the hallmark of most AMPs.Regardless of their final target of killing, AMPs must interact with thebacterial cytoplasmic membrane, and amphipathicity is integral to suchinteractions. The cationic region facilitates electrostatically drivenadsorption to anionic bacterial membranes and imparts some measure ofselectivity, since mammalian cell membranes are largely zwitterionic.The hydrophobic region provides an additional driving force forincorporation of the AMP into the lipid bilayer. The precise nature ofAMP-membrane interactions remains controversial and actively debated; avariety of mechanisms have been proposed, including the carpet,barrel-stave pore, toroidal pore, and aggregate models.

SUMMARY OF THE INVENTION

In light of the foregoing, it is an object of the present invention toprovide new poly-N-substituted glycine compounds and methods for and/ortherapies relating to their use as antibiotics, thereby improving uponthe prior art and/or overcoming various deficiencies or shortcomingsthereof. It will be understood by those skilled in the art that one ormore aspects of this invention can meet certain objectives, while one ormore other aspects can meet certain other objectives. Each objective maynot apply equally, in all its respects, to every aspect of thisinvention. As such, the following objects can be viewed in thealternative with respect to any one aspect of this invention.

It can be an object of the present invention to provide such compoundshaving minimum inhibitory concentrations in the low micromolar rangeagainst both Gram-positive and Gram-negative bacteria, with lowermammalian cytotoxicities and negligible hemolysis, at suchconcentrations, as compared to compounds of the prior art.

It can be another object of the present invention to provide suchcompounds, variable by residue sequence and/or N-substituent, so as toaffect hydrophobicity and/or amphipathicity and/or to enhanceselectivity.

It can be another object of the present invention alone or inconjunction with one or more of the preceding objectives, to provide anew class of N-alkylated peptoids, providing such potencies andselectivities at monomer numbers and peptoid lengths shorter thanpreviously available.

Other objects, features, benefits and advantages of the presentinvention will be apparent from this summary and its descriptions ofcertain embodiments, and will be readily apparent to those skilled inthe art having knowledge of various peptidomimetic compounds and theirsyntheses. Such objects, features, benefits and advantages will beapparent from the above as taken into conjunction with the accompanyingexamples, data, figures and all reasonable inferences to be drawntherefrom, alone or with consideration of the references incorporatedherein.

In part, the present invention can be directed to a poly-N-substitutedglycine antibiotic compound of a formula

In such a compound, A can be selected from H and a terminal N-alkylsubstituted glycine residue, where such an alkyl substituent can beselected from about C₄ to about C₂₀ linear, branched and cyclic alkylmoieties; n can be an integer selected from 1-3; B can be selected fromNH₂, and one and two N-substituted glycine residues, such N-substituentsas can be independently selected from α-amino acid side chain moietiesand structural/functional analogs thereof; and X, Y and Z can also beindependently selected from N-substituted glycine residues, suchN-substituents as can be independently selected from α-amino acid sidechain moieties and structural/functional analogs thereof and prolineresidues. As described elsewhere herein, such X—Y—Z periodicity canprovide such a compound a certain amphipathicity. As would be understoodby those skilled in the art made aware of this invention, suchstructural and/or functional analogy can be considered in the context ofany such α-amino acid side chain, N-substituent and/or a sequence ofsuch N-substituted glycine residues, such structure and/or functionincluding but not limited to charge, chirality, hydrophobicity,amphipathicity, helical structure and facial organization. Such analogsinclude, without limitation, carbon homologs of such side chain—suchhomologs as would be understood in the art, including but not limited toplus or minus 1 or 2 or more methylene and/or methyl groups.

Regardless, in certain embodiments A can be H, and B can be selectedfrom one or two N-substituted glycine residues, such a selection as canreduce the hydrophobicity of such a compound, as compared to compoundsof 3-fold periodicity. In certain such embodiments, X can be an N_(Lys)residue; n can be 2-3; and B can be two N-substituted glycine residues.Without limitation, such a compound can be of a formula

In various other embodiments, regardless of identity of A, X and B, atleast one of Y and Z can be a proline residue. In certain suchembodiments, X, Y and Z can be proline residues.

In certain other embodiments, A can be a terminal N-alkyl substitutedglycine residue, with such an alkyl substituent as can be selected fromabout C₆ to about C₁₈ linear alkyl moieties. Regardless, B can be NH₂,and n can be selected from 1 and 2. In certain such embodiments, A canbe a terminal N-alkyl substituted glycine residue, with an alkylsubstituent selected from about C₆ to about C₁₈ linear alkyl moieties.Regardless, B can be an N_(Lys) residue, and n can be 1.

In part, this invention can also be directed to a poly-N-substitutedglycine antibiotic compound of a formula

wherein n can be selected from 2 and 3; and Y, Z, Y′ and Z′ can beindependently selected from N-substituted glycine residues, where suchsubstituents can be independently selected from α-amino acid side chainmoieties and carbon homologs thereof. Such Y′ and Z′ residues can beselected to provide such compound reduced hydrophobicity as compared toa compound of 3-fold periodicity. In certain such embodiments, at leastone of X and Y can be a proline residue. Regardless, n can be selectedfrom 2 and 3, and Y′ can be an N_(Lys) residue. In certain suchembodiments, one or both X and Y can be proline residues. Withoutlimitation, such a compound with reduced hydrophobicity can be of aformula

In part, this invention can also be directed to a poly-N-alkylsubstituted glycine antibiotic compound of a formula

wherein B can be selected from NH₂ and X′; X, Y, Z and X′ can beindependently selected from N-substituted glycine residues, where suchsubstituents can be independently selected from α-amino acid side chainmoieties and carbon homologs thereof; n can be an integer selected from1 and 2; and R can be an N-alkyl substituent of such a glycine residue,as can be selected from about C₄ to about C₂₀ linear, branched andcyclic alkyl moieties. In certain embodiments, n can be 2, and B can beNH₂. In certain other embodiments, n can be 1, and B can be X′.Accordingly, one or both of X and X′ can be N_(Lys) residues.Regardless, an alkyl substituent can be selected from about C₆ to aboutC₁₈ linear, branched and cyclic alkyl moieties, and X and X′ can beN_(Lys) residues. Without limitation, such a compound can be of aformula

H—N_(tridec)—N_(Lys)—N_(spe)—N_(spe)—N_(Lys)—NH₂.

In part, the present invention can be directed to a poly-N-substitutedglycine antibiotic compound comprising an N-terminus selected from H andan N-alkyl substituted glycine residue, where such an alkyl substituentcan be selected from about C₄ to about C₂₀ linear, branched and cyclicalkyl moieties; a C-terminus selected from NH₂, one and twoN-substituted glycine residues, such N-substituents as can beindependently selected from α-amino acid side chain moieties andstructural/functional analogs thereof; and 2 to about 15 monomericresidues between the N- and C-termini, each such residue as can beindependently selected from proline residues and N-substituted glycineresidues, said N-substituents independently selected from α-amino acidside chain moieties and structural/functional analogs thereof. Asillustrated herein and as distinguished over the prior art, suchmonomers can be selected to provide such a compound a non-periodicsequence of monomers. As would be understood by those skilled in the artmade aware of this invention, such structural and/or functional analogycan be considered in the context of any such α-amino acid side chain,N-substituent and/or a sequence of such N-substituted glycine residues,such structure and/or function including but not limited to charge,chirality, hydrophobicity, amphipathicity, helical structure and facialorganization. Such analogs include, without limitation, carbon homologsof such side chain—such homologs as would be understood by those skilledin the art, including but not limited to plus or minus 1 or 2 or moremethylene and/or methyl groups.

In certain embodiments, the N-terminus of such a compound can be H; andthe C-terminus can be selected from said one and two N-substitutedglycine residues. Regardless, such a compound can comprise 2 to about 5(X—Y—Z) non-periodic trimers. In certain such embodiments, at least oneof X, Y and Z in each of the trimers can be selected to interrupt 3-foldperiodicity. Without limitation, at least one X in at least one saidtrimer can be an N_(Lys) residue. In certain such embodiments, at leastone of Y and Z in at least one such trimer can be a proline residue. Inother embodiments, the monomeric residues can comprise at least twonon-consecutive of the same or repeat trimers, with at least one suchresidue therebetween to interrupt periodicity. In certain suchembodiments, at least one X in at least one such trimer can be anN_(Lys) residue, and at least one of Y and Z in at least one said trimercan be a proline residue.

In various other non-limiting embodiments, the N-terminus of such acompound can be an N-alkyl substituted glycine residue, with an alkylsubstituent selected from about C₆ to about C₁₈ linear alkyl moieties.Regardless, such a compound can comprise 2 to about 5 (X—Y—Z)non-periodic trimers. In certain such embodiments, at least one of X, Yand Z in each of the trimers can be selected to interrupt 3-foldperiodicity. In certain other embodiments, the monomeric residues cancomprise at least two non-consecutive of the same or repeat trimers,with at least one residue therebetween to interrupt periodicity. Incertain such embodiments, at least one X in at least one said trimer canbe an N_(Lys) residue, and at least one of Y and Z in at least one saidtrimer can be a proline residue.

In part, the present invention can also be directed to one or moreantimicrobial peptoid compositions comprising one or more of thepoly-N-substituted glycine compounds of this invention. Such compoundsas can optionally comprise one or more antimicrobial peptides and/orpeptidomimetic compounds now or hereafter known in the art. Accordingly,this invention can be directed to a range of pharmaceutical compositionscomprising one or more of the present polypeptoid/ampetoid compounds,optionally with an antimicrobial component of the prior art, and apharmaceutically-acceptable carrier. Such compositions can be preparedand/or formulated as would be understood by those skilled in the artmade aware of this invention. Regardless, as illustrated below, any ofthe present polypeptoid/ampetoid compounds and/or related compositionscan be used alone or in combination, whether administered together orsequentially, in conjunction with one or more bacteria or microbialtreatment methodologies. Without limitation, such a method can compriseproviding one or more such poly-N-substituted glycine compounds and/orrelated compositions; and administering such compound(s)/composition(s)and/or contacting bacteria therewith. As would be understood by thoseskilled in the art, such administration can be in vitro or in vivo,using techniques of the sort described herein or straight-forwardmodifications thereof, such modifications as would also be known tothose skilled in the art and made aware of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Peptoid monomer side chain structures, with full and shorthandnames.

FIG. 2. Cytotoxicity data for selected peptoids and comparator peptidesagainst A549 lung epithelial cells.

FIGS. 3A-B. CD spectra of (A) variants of 1 and (B) variants of 2 in 10mM Tris buffer, pH 7.4.

FIGS. 4A-B. CD spectra of (A) variants of 1 and (B) variants of 2 in 5mM POPC/cholesterol (1:1) SUVs suspended in 10 mM Tris buffer, pH 7.4.

FIGS. 5A-B. CD spectra of (A) variants of 1 and (B) variants of 2 in 5mM POPE/POPG (7:3) SUVs suspended in 10 mM aqueous Tris buffer, pH 7.4.

FIG. 6A-C. X-ray reflectivity data (A) and corresponding fit for DPPGmonolayer before (circles; cartoon inset, top (B)) and after (squares;cartoon inset, bottom (C)) peptoid 1 was injected into the subphasebeneath the monolayer.

FIG. 7. In vivo efficacy of ampetoids in a mouse peritoneal injectionmodel. Five or six mice were included in each study group for eachexperiment; the bacterial colony counts from each of two plates permouse are shown in the dot plot. The horizontal line in each grouprepresents the geometric mean of the population.

FIG. 8. A schematic illustration of the sub-monomer synthetic protocolfor polypeptoids. Steps 2 and 3 are simply repeated for the addition ofeach monomer unit. Once the full polypeptoid has been synthesized, it iscleaved off the resin with trifluoroacetic acid and purified byreversed-phase HPLC.

FIG. 9: Schematics of ameptoid variants and side chain structures. Byconvention, the N-terminus is on the left and the C-terminus is on theright. Note that the schematics shown here are for visualizationpurposes only and not intended to imply the actual folding behavior ofeach molecule. Points on the triangular helicies that have no marker areNspe monomers.

FIGS. 10A-C: CD spectra of ampetoid register and sequence variants.Ampetoid concentrations were 60 μM. (A) CD in 10 mM Tris buffer (pH7.4). (B) CD spectra in 10 mM Tris buffer with 5 mM erythrocyte-mimeticPOPC:cholesterol (2:1) SUVs. (C) CD in 10 mM Tris buffer with 5 mMbacteria-mimetic POPE:PEPG (3:7) SUVs.

FIGS. 11A-B: CD spectra of net charge variants in (A) 10 mM Tris bufferand (B) same buffer with 5 mM POPC/cholesterol 2:1 SUVs. Ampetoidconcentration is 60 μM.

FIGS. 12A-C: CD spectra of length variants in (A) 10 mM Tris buffer and(B) same buffer with 5 mM POPC/cholesterol 2:1 SUVs and (C) same bufferwith 5 mM POPE/POPG 3:7 SUVs. Ampetoid concentration is 60 μM.

FIGS. 13A-C: CD spectra of proline variants in (A) 10 mM Tris buffer and(B) same buffer with 5 mM POPC/cholesterol 2:1 SUVs and (C) same bufferwith 5 mM POPE/POPG 3:7 SUVs. Ampetoid concentration is 60 μM.

FIGS. 14A-C: CD spectra of ampetoids containing achiral monomers in (A)10 mM Tris buffer and (B) same buffer with 5 mM POPC/cholesterol 2:1SUVs and (C) same buffer with 5 mM POPE/POPG 3:7 SUVs. Ampetoidconcentration was 60 μM.

FIGS. 15A-C: CD spectra of ampetoids containing opposite chiralitymonomers in (A) 10 mM Tris buffer and (B) same buffer with 5 mMPOPC/cholesterol 2:1 SUVs and (C) same buffer with 5 mM POPE/POPG 3:7SUVs. Ampetoid concentration was 60 μM.

FIGS. 16A-C: CD spectra of ampetoids containing aliphatic monomers in(A) 10 mM Tris buffer and (B) same buffer with 5 mM POPC/cholesterol 2:1SUVs and (C) same buffer with 5 mM POPE/POPG 3:7 SUVs. Ampetoidconcentration was 60 μM.

FIGS. 17A-B: Comparison of antimicrobial/hemolytic activity profiles forselected ampetoids. Those with the most favorable profiles appear at thelower right portion of each plot.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

As illustrated, the present invention provides a class of helical,cationic, amphipathic, sequence- and/or length-specificpoly-N-substituted glycines (peptoids) with potent and very selectiveantibacterial activity. These molecules demonstrate broad-spectrumantimicrobial activity against a range of pathogens, and are able toeffectively treat a bacterial infection in vivo. It has beendemonstrated that these peptoids are both structurally and functionallyanalogous to antimicrobial peptides (AMPs), the natural molecules.Moreover, by modulating the sequence and side chain functionality, theactivity and selectivity of antimicrobial peptoids can be tuned. Somecompounds have minimum inhibitory concentrations (MICs) in the lowmicromolar range against Gram-positive and Gram-negative bacteria, withlow mammalian cytotoxicity and negligible (<1%) hemolysis at their MICs.These activities are substantially improved over previous antibacterialpeptoids earlier reported, the best of which were much more hemolytictoward human red blood cells. This invention also provides a new classof alkylated antibacterial peptoids, which retain the antimicrobialpotency and selectivity in analogs as short as 5 monomers in length.

With reference to examples 1-9 and FIGS. 1-8, below, peptoids weresynthesized, as widely-known in the art, using the submonomer syntheticmethod described by Zuckermann et al., purified using reverse-phase highperformance liquid chromatography (RP-HPLC), and characterized withelectrospray ionization mass spectrometry (ESI-MS) and circulardichroism (CD) spectroscopy. (See, Zuckermann, R. N., Kerr, J. M., Kent,S. B. H., & Moos, W. H. (1992) J. Am. Chem. Soc., 114, 10646-10647, theentirety of which is incorporated herein by reference.) CD spectrum ofantimicrobial peptoids confirms that they adopt helical structures inboth aqueous buffer and lipid vesicles, such that they possess afacially amphipathic organization of cationic and hydrophobic residues.Antibacterial activity was determined according to Clinical LaboratoryStandards Institute (CLSI) protocols for broth microdilution, andhemolytic activity determined using similar microdilution methods. Theeffect of peptoids on cellular metabolic activity was determined usingthe colorimetric tetrazolium salt-based MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]assayalso using serial microdilution. These data demonstrate broad-spectrumactivity against a variety of clinically relevant pathogens, yet themost selective antimicrobial peptoids do not harm human red blood cellsor mammalian fibroblast cells at concentrations many times theirantibacterial concentrations. Lastly, the ability of two peptoids totreat an infection in vivo using a murine peritoneal injection model wasevaluated. These results show that peptoids can significantly reduce thebacterial counts in the mouse peritoneal cavity over a 24 hour exposureperiod without mouse fatality.

Structure-activity studies of antimicrobial peptoids suggest closeanalogy between their mechanisms of bacterial killing and those of AMPs.Numerous studies of AMPs have found that, in general, increases inhydrophobicity and amphipathicity and decreases in net charge can bias apeptide toward a less selective mechanism. The converse is true, in thatadding cationic charge, and decreasing hydrophobicity or amphipathicitycan lead to a more selective peptide. The structure-activity studiesconducted show that peptoids respond similarly to changes inphysicochemical properties. That is, selective peptoids become lessselective when their sequences are altered to make them morehydrophobic, more amphipathic, or less cationic, and vice versa.Antimicrobial peptoids are thus functional analogs of AMPs.

Specular X-ray reflectivity was used to examine molecular-levelinteractions of antimicrobial peptoids with lipids. Just as selectiveAMPs spend the majority of their time oriented parallel to the plane ofthe membrane surface, antimicrobial peptoids are oriented in anionicDPPG monolayers with 56° between the surface normal and the long axis ofthe peptide—clearly not perpendicular to the membrane, which would beexpected for a transmembrane pore-forming peptide—with clearinteractions between the peptoid and the lipid headgroups. The similarlipid-bound orientations of AMPs and antimicrobial peptoids furthersuggests mechanistic analogy between the two classes of molecules.

The present invention, therefore, provides a novel class of antibioticswhich offers a combination of several desirable properties, includingbut not limited to: 1. Efficacy in reducing bacterial counts in amammalian system over a 24-hour exposure using an intraperitonealinjection mouse model. 2. low-micromolar antibacterial activity againsta broad spectrum of clinically relevant pathogens; 3. selectivity thatis tunable by molecular sequence characteristics, whereby mammaliancells are not harmed at the compounds' MICs; 4. functional analogy tonatural antimicrobial peptides, which implies that, like AMPs,antimicrobial peptoids are not readily susceptible to the development ofbacterial resistance.

The role of hydrophobicity is articulated by studies of antimicrobiallipopeptides, such as polymyxin B and trichogin. These AMPs are composedof a peptide chain with a fatty acid tail at the N-terminus. Forexample, the deacylated version of polymyxin B is much less bactericidalthan the natural lipopeptide. Also, trichogin analogues with varyinglengths of lipids showed greater activity with longer tails, andanalogues with tails shorter than four carbon atoms were found to beinactive. Attachment of fatty tails to AMPs that are not normallyacylated has also been attempted, and in some cases, alkylation ofinactive cationic peptides was sufficient to endow the resultinglipopeptides with antibacterial or antifungal activity. Furthermore,studies by Shai et al. have found that when varying the length of analkyl tail, a threshold of hydrophobicity is reached at which thepeptide is no longer selective. That is, at a point, increasing taillength only increases hemolytic activity without improving antibacterialactivity.

As illustrated herein, the in vitro activities of simple peptoid mimicsof AMPs are strikingly similar to those of AMPs themselves. (See, e.g.,Table 1.) Certain antimicrobial peptoids (“ampetoids”) exhibitbroad-spectrum antimicrobial activity, low hemolysis, minimal effects onmammalian cellular metabolism, and efficacy at treating an infection invivo. Furthermore, conjugation of fatty tails to peptoids can lead toselective, non-natural analogs of AMPs as short as five monomers inlength that are potent against both bacteria and fungi.Structure-activity relationships observed in a library of rationallydesigned ampetoids are wholly analogous to those which describe manyAMPs. Using synchrotron radiation to probe the interactions betweenampetoids and lipid layers, AMP-ampetoid analogy was found to extend tomolecular-level interactions.

TABLE 1Antibacterial and hemolytic activities of ampetoids and AMPs. Peptoidmonomer abbreviations are explained in FIG. 2. For minimum inhibitoryconcentrations (MICs) and hemolytic doses (HDs) reported as “>x”, x =200 μg/mL-thehighest concentration tested (except for pexiganan, which was tested up to 500 μg/mL).Variant HPLC elution class Shorthand name Sequence solvent* Basis 1H-(NLys-Nspe-Nspe)₄-NH₂ 48% 2 H-(Nlys-Nssb-Nspe)₄-NH₂ 39% Chirality1_(enantiomer) H-(Nlys-Nrpe-Nrpe)₄-NH₂ 48% Length 1_(6mer)H-(Nlys-Nspe-Nspe)₂-NH₂ 41% 1_(9mer) H-(Nlys-Nspe-Nspe)₃-NH₂ 46%1_(15mer) H-(Nlys-Nspe-Nspe)₅-NH₂ 51% Hydro- 2-Nsap_(2,5,8,11)H-(Nlys-Nsap-Nspe)₄-NH₂ 48% phobicity 2-Nsna_(6,12)H-(Nlys-Nssb-Nspe-Nlys-Nssb- 47% Nsna)₂-NH₂ 1-Nsna_(6,12)H-(Nlys-Nspe-Nspe-Nlys-Nspe- 53% Nsna)₂-NH₂ 1-Nhis_(6,12)H-(Nlys-Nspe-Nspe-Nlys-Nspe- 37% Nhis)₂-NH₂ 1-Pro₆H-Nlys-Nspe-Nspe-Nlys-Nspe-L- 40% Pro-(Nlys-Nspe-Nspe)₂-NH₂ Charge1-Nglu_(4,10) H-(Nlys-Nspe-Nspe-Nglu-Nspe-  60%^(†) Nspe)₂-NH₂1-Nglu_(1,4,7,10) H-(Nglu-Nspe-Nspe)₄-NH₂  54%^(†) Amphi- 1_(block)H-(Nlys)₄-(Nspe)₈-NH₂ 54% pathicity 2_(scrambled)H-Nlys-Nssb-Nspe-Nssb-Nspe- 42% Nlys-Nspe-Nlys-Nssb-Nssb- Nspe-Nlys-NH₂AMPs pexiganan¹ GIGKFLKKAKKFGKAFVKIL 38% KK (SEQ ID NO: 1)-NH₂ melittin⁵GIGAVLKVLTTGLPALISWIK 54% RKRQQ (SEQ ID NO: 2)-NH₂ B. subtilis MICHD₁₀/HD₅₀ Selectivity ratio E. coli MIC (μM) (μM) (μM)  (SR)^(‡) 3.50.88  21/100 6.0 31 3.9 >120/>120 >39 3.5 0.88 16/86 4.6 2727 >220/>220 >8.1 9.1 1.2 >150/>150 >16 5.5 1.4  3/19 0.55 7.40.95 >120/>120 >16 7.2 0.93   55/>120 7.6 3.3 1.6  4/22 1.2 3.56.9 >110/>110 >31 3.1 1.6   63/>110 20 >110 6.9 19/40<0.17 >219 >219 >110/>110 N/A 6.9 1.7 18/73 2.6 31 15 >120/>120 >39 3.11.6   73/>200 24 1.6 0.78 2/6 1.3 *Percent acetonitrile in water, 0.1%(v/v) trifluoroacetic acid (TFA) at HPLC elution. ^(†)With 10 mMammonium acetate and no TFA, pH 7.0. ^(‡)Selectivity ratio, SR =(HD₁₀)/(E. coli MIC). ^(§)For concentrations reported as “>x”, x = 200μg/mL-the highest concentration tested (except for pexiganan, which wastested up to 500 μg/mL). Initial antibacterial activity and selectivityscreening

An initial set of 15 ampetoid analogs was synthesized to determinewhether peptoids are affected by structural and sequence modificationsin a manner consistent with AMP activities. The designs for ampetoids inthis library were derived from two antibacterial and selectiveamphipathic dodecamers, 1 and 2. Peptoid 1 [H-(Nlys-Nspe-Nspe)₄-NH₂] iscomposed of ⅔ Nspe, the peptoid analog of phenylalanine, and ⅓ Nlys, thepeptoid analog of lysine (see FIG. 1 for the structures ofrepresentative peptoid monomers and corresponding N-substituents).Peptoid 2 [H-(Nlys-Nssb-Nspe)₄-NH₂] contains ⅓ isoleucine-like Nssbmonomers in place of Nspe. The variant sequences were designed to effectchanges in chirality, length, hydrophobicity, charge, andamphipathicity. All of these compounds were tested for antibacterialactivity against representative BSL1 Gram-negative (E. coli JM 109) andGram-positive (B. subtilis BR151) bacterial strains. As an initialmeasure of selectivity, the lytic activity of the peptoids wasdetermined against human erythrocytes. Table 1 summarizes the sequencessynthesized, the solvent composition at RP-HPLC elution as a relativemeasure of molecular hydrophobicity, and antibacterial and hemolyticactivities. Ten of the 15 peptoids exhibit low-micromolar MICs againstboth E. coli and B. subtilis, demonstrating that non-natural peptoidoligomers can be as active as AMPs (MICs for pexiganan—a selective AMPanalog of magainin-2- and the bee-venom AMP melittin are shown in Table1).

A selectivity ratio (SR) for each compound was defined, also shown inTable 1, as the quotient of the 10% hemolytic dose (HD₁₀) and the E.coli MIC. Thus, the SR is an estimate of an ampetoid's tendency to killbacteria rather than mammalian cells. Ampetoid 1 has an SR of 6.0,similar to that of pexiganan (SR=5.8). As expected, melittin (well knownto be cytotoxic) has a low SR of 0.16. Most AMPs have antibacterialactivities in the low-micromolar range; since peptoid 1 has MICs in thatrange, the ampetoid library was primarily expected to yield variantswith increased selectivity. Indeed, six of 13 variants are moreselective than 1 and pexiganan (i.e. SRs>6).

The biocompatibility of selected oligomers with A549 lung epithelialcells was evaluated using the MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]assay.FIG. 2 shows that the 10% metabolic inhibitory dose (ID₁₀) and 50%inhibitory dose (ID₅₀) of these selected peptoids compare very favorablyto that of pexiganan and melittin; ampetoids 1-Pro₆ and 1-Nhis_(6,12),with MICs<7 μM, have ID₁₀'s that are 10 and 20 times that of pexiganan.Interestingly, the relatively non-selective peptoid 1_(15mer) exhibitscytotoxicity no worse than pexiganan.

Structure-activity relationships derived from a library of ampetoids arediscussed below according to the primary physicochemical parameter thatwas altered. As in previous studies, circular dichroism (CD)spectroscopy was used to compare the helicities of the ampetoids bymonitoring the intensity of spectral extrema, particularly at 190 and220 nm. CD was performed in 10 mM aqueous Tris buffer, pH 7.4, alone andin the presence of two types of small unilamellar vesicles (SUVs)composed of model binary lipid mixtures: (1) anionic E. colimembrane-mimetic SUVs [POPE/POPG (7:3 mole ratio)], and (2) zwitterionicerythrocyte membrane-mimetic SUVs [POPC/cholesterol (CH) (1:1 moleratio)].

With regard to chirality, using enantiomeric side chains (Nrpe in placeof Nspe; FIG. 1), a left-handed helical analog of peptoid 1 was created,called 1_(enantiomer), as evidenced by its mirror-image CD spectrum incomparison to that of 1 (FIG. 3A). The activities and selectivities of 1and 1_(enantiomer) are congruent (Table 1).

With regard to length, variants of 1 were created, ranging from 6 to 15monomers, all with the same three-fold sequence repeat and 1:3charge-to-length ratio. The shortened variants of 1, 1_(6mer) and1_(9mer) (approx. 12 Å and 18 Å in length, respectively), are bothactive antimicrobials and are more selective than 1 (Table 1). Incontrast, the lengthened variant of 1, 1_(15mer) (approx. 30 Å inlength), is substantially more hemolytic (SR=0.55) and cytotoxic (FIG.2) than 1, and slightly less antibacterial. The helicities of thesecompounds in buffer are congruent (data not shown).

Hydrophobicity was modulated independently of length by replacinghydrophobic Ile-like Nssb and Phe-like Nspe monomers in 1 and 2 withbulkier and more hydrophobic Nsmb and Nsna, respectively (FIG. 1). Theheightened molecular hydrophobicity of 2-Nsmb_(2,5,8,11) and2-Nsna_(6,12) led to increases in both antibacterial and hemolyticactivities (Table 1). 1-Nsna_(6,12), however, shows no enhancement ofantibacterial activity relative to 1, but is much more hemolytic(SR=1.2).

Two variants of 1 with reduced hydrophobicity were also created. Twoevenly spaced hydrophobic Nspe residues in 1 were replaced by Nhis, anachiral peptoid monomer analog of histidine (FIG. 1) that is polar yetpredominantly uncharged at physiological pH, yielding 1-Nhis_(6,12).This oligomer exhibits substantially decreased hemolysis (SR>31;Table 1) and decreased cytotoxicity (FIG. 2), with only a slightreduction in antibacterial activity against B. subtilis compared to 1. Asecond reduced-hydrophobicity variant, 1-Pro₆, was created by replacingthe Nspe at position 6 with 1-proline. Although proline is well known todestabilize peptide α-helices, 1-Pro₆ and 1 are similarly helical inbuffer (FIG. 3A) since 1-proline is well-accommodated in right-handedtype-1-polyproline-like peptoid helices. 1-Pro₆ (SR=8.9) exhibits lesshemolysis and less cytotoxicity than 1, with similar antibacterialactivity (Table 1, FIG. 2).

With respect to charge, the majority of AMPs are cationic, andreplacement of their basic residues with uncharged or anionic moietiestypically leads to a loss of antibacterial activity. To investigate thisphenomenon in ampetoids, Nlys monomers in 1 were substituted withglutamate-like Nglu (FIG. 1) to create 1-Nglu_(4,10) and1-Nglu_(1,4,7,10). Zwitterionic 1-Nglu_(4,10) has significantly reducedactivity against B. subtilis compared to 1 and is inactive against E.coli (Table 1), likely due to the absence of favorable electrostaticinteractions with anionic bacterial membranes; however, it is quitehemolytic (SR<0.17). The fully anionic variant, 1-Nglu_(1,4,7,10) isdevoid of both antibacterial and hemolytic activity.

To study amphipathicity, a terminally, rather than facially, amphipathicisomer of 1 with block-like architecture (1_(block)) was created, aswell as a scrambled sequence of 2 designed to preclude globalamphipathicity (2_(scrambled)) (Table 1). The terminal segregation ofcationic and hydrophobic residues in the 1_(block) sequence ensures astrongly amphipathic structure independent of the facial organization ofresidues along a helix. 1_(block) is slightly less antibacterial andmore hemolytic than 1 (SR=2.6), although it has the same monomercomposition. The CD spectra of 2_(scrambled) in buffer and lipidenvironments (FIGS. 3B, 4B, 5B) are nearly congruent to those of 2,suggesting 2_(scrambled) forms a structured helix which, due to itsscrambled sequence, has low global amphipathicity relative to 2. Peptoid2_(scrambled) exhibits antibacterial activity, but no hemolysis up to120 μM.

TABLE 2Antibacterial, antifungal, and hemolytic activities of alkylated variants ofpeptoid 1. See FIG. 1 for a guide to monomer structures. HPLC PEPTOIDSEQUENCE ELUTION^(†) 1 H-(Nlys-Nspe-Nspe)₄-NH₂ 48% C5-1H-Npent-(Nlys-Nspe-Nspe)₄-NH₂ 51% C10-1 H-Ndec-(Nlys-Nspe-Nspe)₄-NH₂ 58%1_(9mer) H-(Nlys-Nspe-Nspe)₃-NH₂ 46% C5-1_(9mer)H-Npent-(Nlys-Nspe-Nspe)₃-NH₂ 48% C10-1_(9mer)H-Ndec-(Nlys-Nspe-Nspe)₃-NH₂ 59% 1_(6mer) H-(Nlys-Nspe-Nspe)₂-NH₂ 47%C5-1_(6mer) H-Npent-(Nlys-Nspe-Nspe)₂-NH₂ 49% C10-1_(6mer)H-Ndec-(Nlys-Nspe-Nspe)₂-NH₂ 59% C10-1_(4mer)H-Ndec-Nlys-Nspe-Nspe-Nlys-NH₂ 50% C13-1_(4mer)H-Ntridec-Nlys-Nspe-Nspe-Nlys-NH₂ 57% PexigananGIGKFLKKAKKFGKAFVKILKK (SEQ ID NO: 1)-NH₂ 38% E. coli B. subtilisC. albicans HD₁₀/HD₅₀ MIC (μM) MIC (μM) MIC (μM) (μM) 6.3 1.6 6.3 18/726.3 1.6 6.3 10/40 12.5 1.6 1.6  8/27 25 1.6 15.6  150/>200 16.3 1.0 12.5  65/>200 6.3 3.1 3.1 12/40 27* 27* >100 >220/>220 >200 3.1100 >200/>200 6.3 1.6 6.25 30/80 >100 6.3 100 >200/>200 12.5 1.6 12.5 70/200 12.5 1.6 50   70/>200 *All assays were performed incation-adjusted broth except where denoted by an asterisk. ^(†)HPLCelution is given as a measure of hydrophobicity and defined as percentacetonitrile in H₂O (C18 column, conducted in the presence of 0.1% TFA,pH 1).

In order to test the effects of alkylation on antimicrobial peptoids, aseries of alkylated variants of 1 was synthesized, systematicallyvarying both the alkyl tail length and the length of the peptoid chain.In general, the length of the peptoid chain was decreased in increments3 monomers (one full helical turn) from the original sequence of 1.However, based on the report of active ultrashort AMPs containing twopositive charges, an additional Nlys monomer was retained at theshortest length. Alkyl tails were incorporated via the submonomerpeptoid synthetic protocol (using the appropriate alkylamine for thesubstitution step) as the side chain of the N-terminal peptoid residue.All peptoids were amidated at the C-terminus. Table 2 lists theshorthand names and sequences of the alkylated peptoids investigated, aswell as the activities of these alkylated variants. Interestingly, itshould be noted that C13-1_(4mer)—a compound roughly half the molecularweight of 1—exhibited antibacterial activity comparable to 1 and was infact found to be more selective against erythrocytes.

Additionally, in order to characterize possible antifungal activity, alibrary of alkylated compounds was tested against C. albicans, arepresentative fungal strain. In several instances, compounds such asC10˜1_(6mer) and C13˜1_(4mer) were found to have potent and selectiveantifungal activity.

Whereas the preceding generation of molecules was designed to elucidatestructure function relationships of specific molecular parameters(chirality, length, hydrophobicity, etc.), another generation compoundswere designed explicitly to explore effects on selectivity.Antimicrobial activity of these compounds was tested against bacterialstrains E. coli (ATTC 35218) and B. subtilis (ATTC 6633) incation-adjusted MHB using the microdilution protocols previouslydescribed. The sequences, antimicrobial activities, hemolyticactivities, and metabolic inhibitory concentrations against NIH 3T3mouse fibroblast cells are summarized in Table 3 and the rationale oftheir design is described below.

TABLE 3Sequences and activities of second-generation peptoids designed to explore selectivity. Variant class Shorthand name Sequence Basis 1H-(Nlys-Nspe-Nspe)₄-NH₂ Register variants 1B H-(Nspe-Nlys-Nspe)₄-NH₂ 1CH-(Nspe-Nspe-Nlys)₄-NH₂ Proline variants 1-Pro₃H-(Nlys-Nspe-L-Pro)-(Nlys-Nspe-Nspe)₃-NH₂ 1-Pro₆H-Nlys-Nspe-Nspe-Nlys-Nspe-L-Pro-(Nlys-Nspe-Nspe)₂-NH₂ 1-Pro₉H-(Nlys-Nspe-Nspe)₂-Nlys-Nspe-L-Pro-Nlys-Nspe-Nspe-NH₂ 1-Pro_(3,9)H-Nlys-Nspe-L-Pro-Nlys-Nspe-Nspe-Nlys-Nspe-L-Pro- Nlys-Nspe-Nspe-NH₂Nhis variants 1-Nhis₆H-Nlys-Nspe-Nspe-Nlys-Nspe-Nhis-(Nlys-Nspe-Nspe)₂-NH₂ 1-Nhis_(6,12)H-(Nlys-Nspe-Nspe-Nlys-Nspe-Nhis)₂-NH₂ 1-Nhis_(3,6,9,12)H-(Nlys-Nspe-Nhis)₃-NH₂ Achiral variants 1_(achiral)H-(Nlys-Npm-Npm)₄-NH₂ 1_(achiral)-Nspe₁₂H-(Nlys-Npm-Npm)₃-Nlys-Npm-Nspe-NH₂ 1-Npm_(2,5,8,11)H-(Nlys-Npm-Nspe)₄-NH₂ 1-Npm_(2,3,8,9)H-(Nlys-Npm-Npm-Nlys-Nspe-Nspe)₂-NH₂ Nsdp variants 1-Nsdp_(all)H-(Nlys-Nsmb-Nsmb)₄-NH₂ 1-Nsdp_(2,5,8,11) H-(Nlys-Nsmb-Nspe)₄-NH₂ Charge1-Nlys_(5,11) H-(Nlys-Nspe-Nspe-Nlys-Nlys-Nspe)₂-NH₂ distribution1B_(12mer)-Nlys_(4,10) H-(Nspe-Nlys-Nspe-Nlys-Nlys-Nspe)₂-NH₂ variants1B_(15mer)-Nlys_(4,10)H-(Nspe-Nlys-Nspe-Nlys-Nlys-Nspe)₂-Nspe-Nlys-Nspe-NH₂1B_(12mer)-Nlys_(4,6,10)H-Nspe-Nlys-Nspe-Nlys-Nlys-Nlys-Nspe-Nlys-Nspe-Nlys- Nlys-Nspe-NH₂1B_(15mer)-Nlys_(4,6,10)H-Nspe-Nlys-Nspe-Nlys-Nlys-Nlys-Nspe-Nlys-Nspe-Nlys-Nlys-Nspe-Nspe-Nlys-Nspe-NH₂ AMP comparator pexigananGIGKFLKKAKKFGKAFVKILKK (SEQ ID NO: 1)-NH₂ E. coli B. subtilis HD₁₀/HD₅₀MIC (μM) MIC (μM) (μM) ID₁₀ID₅₀ ^(‡) 6.3 1.6  21/100 5.1/1.4 6.3 1.6  40/>100 4.9/2.5 6.3 1.6   25/>100 5.6/2.3 12.5 1.6   74/>200 4.8/12 12.5 1.6   83/>200 8.2/18  12.5 1.6  165/>200 9.8/24  50 1.6 >200/>20031/71 25 1.6  170/>200 — >50 1.6 >110/>110 >100/>100 >100 100 >200/>200  72/>100 — — >100/>100 5.1/16  25 1.6  164/>200 — 6.3 1.6   87/>2003.5/6.8 6.3 1.6   68/>200 — 50 0.78 >200/>200 6.6/64  12.5 0.78 111/>200 10/20 50 1.6 >100/>100 23/85 50 0.78 >200/>200 — 500.78 >200/>200 — >100 1.6 >200/>200 — >100 0.78 >200/>200 — 12.5 1.6  70/>200 1.9/9  

Without limitation, two molecules in Table 1 found to be extraordinaryselective (1-Pro₆ and 1-Nhis_(6,12)) were used to derive several newsequences. Since 1-Pro₆ was found to be highly antimicrobial but lesshemolytic than 1, two isomerically related compounds (1-Pro₃, 1-Pro₉)were created to explore the effect of proline residue position onactivity and selectivity. One variant with prolines in both the thirdand ninth position (1-Pro_(3,9)) was also designed to evaluate theeffect of increasing the number of proline residues on the activityprofile. A second pair of molecules (1-Nhis₆ and 1-Nhis_(3,6,9,12)) wascreated to explore the effect of modulating the number of Nhis residueswithin the sequence.

The effects of hydrophobicity on activity and selectivity was furtherexplored with an additional set of molecules described in Table 3.Several sequences were designed with replacement of selected Nsperesidues by achiral Npm residues (see FIG. 1). Two sequences containedfour Npm residues either aligned on one face (1-Npm_(2,5,8,11)) ordistributed on two faces (1-Npm_(2,3,8,9)) of the peptoid. Additionally,the molecule (1_(achiral)-Nspe₁₂) with all Nspe replaced with Npm exceptfor the 12^(th) position was designed to be less hydrophobic, yet stillsomewhat helical; it has been shown that inclusion of a chiral residueat the C-terminus promotes helicity. Lastly, an all achiral version of1, 1_(achiral), contains all Nspe residues substituted with Npm.

The hydrophobic side chain Nsdp, which is isomerically related to Nsmb(FIG. 1), was incorporated into two additional sequences aimed atfurther investigating the use of aliphatic side chains. 1-Nsdp_(all) hasall Nspe monomers replace with Nsdp, and 1-Nsdp_(2,5,8,11) displays fourNsdp monomers on one face.

A final family of sequences was designed to evaluate the effect ofsequence register, charge distribution, and length on activity andselectivity. Compounds 1B and 1C are isomers of 1 in which the sequenceregister was changed to preclude the presence of terminal charges(Nspe-Nlys-Nspe) or exhibit a C-terminal charge (Nspe-Nspe-Nlys),respectively. Several compounds were also created with increased numberof charges (i.e. decreased hydrophobicity) distributed over multiplefaces of the molecule. Compound I-Nlys_(5,11) exhibits the sequence of 1with the two Nspe monomers at positions 5 and 11 replaced with Nlysresulting in two additional charges. Other variants include a 1Bregister sequence analogously substituted with Nlys and positions 4 and10 as both a 12mer (1B_(12mer)-Nlys_(4,10)) and a 15mer(1B_(15mer)-Nlys_(4,10)). Similarly, molecules with additional Nlysmonomers distributed on three faces (1B_(12mer)-Nlys_(4,6,10) and1B_(15mer)—Nlys_(4,6,10)) were also created.

X-Ray Reflectivity Studies of Ampetoid Orientation in Lipid Layers

Liquid (aqueous buffer) surface specular X-ray reflectivity (XR) studieswere conducted using synchrotron radiation to investigate the membraneorientation and depth of penetration of ampetoid 1 in a model lipidlayer which mimics the outer leaflet of the cell membrane (FIG. 6).X-rays reflected off of the monolayer yield an electron density profileperpendicular to the interface, allowing determination of the layerthickness and the presence and orientation of added molecules. Theexperimental data are represented as a stack of slabs, each with auniform thickness, electron density, and interface roughness.

XR data for a pure DPPG (anionic) film (FIG. 6—circles) fit well with atwo-slab model, yielding a hydrocarbon tail density (ρ_(t)/ρ_(s)) of0.99 and a hydrocarbon tail slab thickness (L₁) of 17.9 Å, as well as aheadgroup electron density (ρ_(h)/ρ_(s)) of 1.54 and headgroup slabthickness (L₂) of 5.7 Å. These data are in good agreement with previousDPPG monolayer X-ray work. The XR profile changed dramatically afterpeptoid 1 was introduced (FIG. 6—squares), and fit a four-slab model.According to this fit, the first slab (ρ_(t)/ρ_(s)=0.96, L₁=12.1 Å)corresponds to the lipid tails without 1, the second slab(ρ_(t+p)/ρ_(s)=1.05, L₂=2.8 Å) to the tails region with partialinsertion of 1, the third slab (ρ_(h+p)/ρ_(s)=1.33, L₂=7.0 Å) to thelipid headgroup region with 1 fully inserted, and the fourth slab(ρ_(p)/ρ_(s)=1.16, L₂=3.6 Å) to 1 alone, protruding beyond the DPPGheadgroups. This electron density profile is consistent with insertionof 1 through the lipid headgroups and partially into the lipid tailregion. Furthermore, assuming that 1 retains its helical structure inmodel lipid monolayers, the data suggest that 1 inserts at an angle ofapproximately 56° between the interface normal and the long helical axisof the peptoid.

TABLE 4 Broad-spectrum antibacterial activity of pexiganan and selectedampetoids against BSL2 and BSL3 pathogenic bacteria. MICs (μM)1_(achiral)- 1- Ntridec- Strain Gram pexiganan 1 1-Pro₆ 1-Pro₉1_(achiral) Nspe_(2,5,8,11) Nsmb_(2,5,8,11) 1_(4mer) Proteus vulgarisATCC 49132 − 12.9 17.6 18.2 36.5 18.7 36.3 19.0 38.3 Pseudomonasaeruginosa ATCC 27853 − 1.6 4.4 18.2 18.2 9.4 36.3 9.5 9.6 Proteusmirabilis − >51.7 >70.4 >72.9 >72.9 >75 >72.6 >76.0 153.3 ATCC 35659Klebsiella pneumoniae ATCC 33495 − 3.2 8.8 9.1 9.1 4.7 9.1 4.8 9.6Enterobacter aerogenes ATCC 35029 − 12.9 8.8 72.9 36.5 75.0 36.319.0 >153.3 Escherichia coli ATCC 25922 − 3.2 2.2 9.1 4.6 9.4 4.5 4.89.6 Serratia marcescens ATCC 13880 − >51.770.4 >72.9 >72.9 >75 >72.6 >76.0 153.3 Staphylococcus aureus ATCC29213 + 12.9 2.2 4.6 9.1 4.7 4.5 4.8 4.8 Staphylococcus aureus VAN1* +6.5 2.2 4.6 4.6 4.7 4.5 4.8 4.8 Staphylococcus aureus VAN2* + 3.2 2.24.6 4.6 4.7 4.5 4.8 4.8 Staphylococcus aureus NRS100 (COL) + 6.5 2.2 4.64.6 4.7 4.5 4.8 4.8 Staphylococcus aureus NRS119 + 25.8 2.2 9.1 9.1 9.49.1 9.5 4.8 Staphylococcus aureus NRS120 + 25.8 4.4 9.1 9.1 9.4 9.1 9.54.8 Enterococcus faecalis ATCC 29212 + 12.9 2.2 4.6 4.6 9.4 4.5 4.8 9.6Enterococcus faecalis 99* + 51.7 4.4 36.5 36.5 37.5 36.3 38.0 19.2Enterococcus faecium 106* + 1.6 2.2 2.3 2.3 2.3 2.3 2.4 4.8 *vancomycinresistant

XR data for a pure DPPG (anionic) film (FIG. 6A—circles; cartoon inset,top (B)) fit well with a two-slab model, yielding a hydrocarbon taildensity (ρ_(t)/ρ_(s)) of 0.99 and a hydrocarbon tail slab thickness (L₁)of 17.9 Å, as well as a headgroup electron density (ρ_(h)/ρ_(s)) of 1.54and headgroup slab thickness (L₂) of 5.7 Å. These data are in goodagreement with previous DPPG monolayer X-ray work. The XR profilechanged dramatically after peptoid 1 was introduced (FIG. 6B—squares;cartoon inset, bottom (C)), and fit a four-slab model. According to thisfit, the first slab (ρ_(t)/ρ_(s)=0.96, L₁=12.1 Å) corresponds to thelipid tails without 1, the second slab (ρ_(t+p)/ρ_(s)=1.05, L₂=2.8 Å) tothe tails region with partial insertion of 1, the third slab(ρ_(h+p)/ρ_(s)=1.33, L₂=7.0 Å) to the lipid headgroup region with 1fully inserted, and the fourth slab (ρ_(p)/ρ_(s)=1.16, L₂=3.6 Å) to 1alone, protruding beyond the DPPG headgroups. This electron densityprofile is consistent with insertion of 1 through the lipid headgroupsand partially into the lipid tail region. Furthermore, assuming that 1retains its helical structure in model lipid monolayers, the datasuggest that 1 inserts at an angle of approximately 56° between theinterface normal and the long helical axis of the peptoid.

Using a murine peritoneal injection mouse model, the ability of peptoidsto treat infection was evaluated in vivo. The results from threeseparate studies of peptoid 1 and two of peptoid 1-Nhis_(6,12) againstsaline treated controls are shown in FIG. 7. The horizontal linerepresents the geometric mean of each population.

Previous work has shown that peptoids can be used to create compoundswith antimicrobial activities similar to AMPs. In this study, the facilesynthesis and high propensity for helix formation of peptoids wasexploited to create and study a library of ampetoid variants thatsuggests functional and mechanistic analogy between ampetoids and AMPsand that demonstrates ampetoids' potential for development intoclinically useful antibiotics.

The equivalent activities of 1 and 1_(enantiomer) demonstrate thatampetoid mechanism is not dependent on overall handedness nor onstereospecific interactions with receptors or enzymes, an attributewhich has also been observed for many AMPs. Evidence that ampetoidsinteract with and insert into membranes is provided by X-rayreflectivity studies (FIG. 6). Furthermore, the depth of insertion ofpeptoid 1—through the headgroups and partially into the lipidtails—demonstrates that 1 interacts simultaneously with hydrophobic andhydrophilic lipid moieties; thus, as with AMPs, the amphipathicstructure of 1 is integral to its interactions with membranes. Theorientation of 1 at an angle of ˜56° to the interface normal suggeststhat 1 does not operate through a barrel-stave mechanism, since thatwould require a transmembrane configuration. Although it cannot beconcluded that 1 exhibits identical mechanistic behavior to natural,α-helical antimicrobial peptides, these X-ray results demonstrateampetoid-lipid interactions consistent with those seen for AMPs such aspexiganan and LL-37.

Ultimately, whether ampetoid activity also adheres to trends relatingstructure and function was determined in a manner analogous to AMPs, andphysicochemical properties of selective ampetoids were found consistentwith those of selective AMPs and non-selective peptoids exhibit closesimilarities to cytotoxic peptides. Numerous structure-activity studiesof a wide variety of AMPs have delineated the physicochemicalcharacteristics that give rise to selective antibacterial activity ornon-selective cytotoxicity. Regardless of structural class (i.e.α-helix, β-sheet, loop, or extended), non-selective AMPs typically (1)are very hydrophobic, such that their interactions with membranes aregoverned primarily by the hydrophobic effect, and (2) have awell-defined amphipathic structure. In contrast, the antibacterialactivity of selective AMPs is dependent on (1) high net cationic charge(although excessive cationic charge can also lead to hemolytic activity)and (2) only moderate hydrophobicity. Perhaps counterintuitively, awell-defined amphipathic structure is not necessary for selectiveantimicrobial activity; destabilization of AMP secondary structure oftenleads to improvements in selectivity.

The ampetoids 1-Nglu_(4,10), 1_(15mer), 1-Nsna_(6,12), and 1_(block) areall less selective than peptoid 1 (SRs<6.0) (Table 1). Consistent withproperties of non-selective AMPs, these compounds are all either morehydrophobic (according to RP-HPLC elution time) and/or less charged(1-Nglu_(4,10)) than 1. Furthermore, they are all as (or more) helicalthan 1 in erythrocyte-mimetic POPC/CH SUVs (FIG. 4), indicative of theirwell-defined membrane-bound amphipathic structures in that lipidenvironment.

In contrast, 1_(9mer), 1-Nhis_(6,12), and 1-Pro₆ all have antibacterialactivities comparable to 1, but enhanced selectivities (SRs>6.0). Thesepeptoids are all more hydrophilic than 1, and all have a net charge ofat least +3. Thus, ampetoids are selectively active provided they have anet positive charge and are sufficiently but not excessivelyhydrophobic, a phenomenon that is wholly consistent with observations ofselective AMPs. Additionally, retention of antibacterial activity andenhancement of selectivity in the shortened 1_(9mer) is analogous tobehavior observed in studies of truncated AMPs.

In general, the effect of length on ampetoid potency and selectivity canlargely be attributed to differences in hydrophobicity (RP-HPLCretention time, Table 1), which increases proportionally with length.Since the 12mer 1 is more antibacterial than longer and shorter analogs,these results suggest the existence of an optimal hydrophobicity atwhich antibacterial activity is maximized; added hydrophobicityincreases only hemolytic activity. This conclusion is also supported bythe hydrophobicity variants, since moderately hydrophobic2-Nsap_(2,5,8,11) and 2-Nsna_(6,12) are both more antibacterial and morehemolytic than 2, while the strongly hydrophobic 1-Nsna_(6,12) (Table 1)shows enhancement of hemolytic but not antibacterial activity relativeto 1. These results are in agreement with observations of AMPs and theirvariants.

Although they are more hemolytic than 2, peptoids 2-Nsna_(6,12) and2-Nsap_(2,5,8,11) are still more selective (SRs of 7.6 and 16,respectively) than 1, and their physicochemical properties areconsistent with this finding. They are highly charged (+4), moderatelyhydrophobic, and exhibit CD spectra consistent with low helicity (FIG.3B, 4B, 5B). The CD spectrum of 2-Nsna_(6,12) exhibits red-shiftedextrema, suggesting a destabilized secondary structure. Thus, thesevariants of 2 all have poorly defined amphipathic structures.

Indeed, results on the whole suggest that, consistent with AMPs, ahighly amphipathic structure is required for hemolytic activity, but notfor antibacterial activity. CD spectra of ampetoids in POPE/POPG SUVs(FIG. 5) show that the extent of helicity in this bacteria-mimeticsystem is poorly correlated with antimicrobial activity. 1-Nsna_(6,12)for example, is less helical than 1 (FIG. 5A), but the two compoundshave similar antibacterial potencies (Table 1). In contrast,2-Nsap_(2,5,8,11) is similarly helical to 2 (FIG. 5B), yet2-Nsmb_(2,5,8,11) is much more antibacterial (Table 1).

CD spectra in erythrocyte-mimetic POPC/CH SUVs (FIG. 4) do, however,reveal a correlation between hemolytic activity and helicity.1-Nsna_(6,12), and 1-Nglu_(4,10) are the most hemolytic ampetoids (Table1), and are the most helical in POPC/CH SUVs (FIG. 4A). 1_(15mer) isalso very hemolytic and as helical as 1 in POPC/CH. Conversely,1-Nhis_(6,12), 2,2-Nsmb_(6,12), and 2_(scrambled) are less hemolytic(Table 1) and less helical (FIG. 4) than 1. Thus, for these cationic,facially amphipathic compounds, it appears that defined helicalstructure, which leads to clean segregation between cationic andhydrophobic groups, is important for hemolytic activity. Further insightis provided by 1_(block), which has a well-defined terminallyamphipathic structure independent of its helicity; 1_(block) ishemolytic (SR=2.6). Together, these results suggest that helicity isimportant only as a means to organize an amphipathic structure, whichfacilitates oligomerization and a bias toward a non-selective mechanism.Based on this conclusion, a helical but poorly amphipathic compound suchas 2_(scrambled) should be antimicrobial, but selective; indeed,2_(scrambled) kills bacteria, but exhibits no hemolysis up to 120 μM.

Results also indicate that hydrophobicity is relevant to hemolyticactivity, and comparison of 1-Pro₆ and 1 establishes this relationshipmore concretely. These ampetoids have similar sequences with the samenet charge and yield nearly congruent CD spectra (FIG. 3A). Thissuggests that their helical content, and thus their resultantamphipathicity, are similar. However, the Nspe→Pro substitution yields areduction in hydrophobicity, according to RP-HPLC elution time (Table1). This single difference is reflected in the lower hemolytic andcytotoxic activities of 1-Pro₆ compared to 1, and reveals clearly thedependence of selectivity on hydrophobicity in a case where all otherparameters are held constant.

The results in Table 3 provide further insight into how molecularstructural features modulate peptoid activity and selectivity. Whileantimicrobial activity does not change with respect to proline position,cytotoxicity is decreased as the proline is moved closer to theC-terminus. It is possible that proline provides structural stability atthe C-terminus that makes it less harmful to mammalian cells while notaffecting its ability to kill bacteria. Increasing the number of Nhisresidues decreased both the antimicrobial activity as well as thecytotoxicity. Interesting, 1-Nhis_(6,12), the basis sequence for thisfamily, was found to be far less active against E. coli ATCC 35218 thanthe strain originally tested (JM109).

The family of compounds containing varying amounts of achiral residuesshows that while the positions of the achiral residues does notsignificantly affect antimicrobial activity, it may have an impact onselectivity (Table 3,1-Npm_(2,5,8,11) vs. 1-Npm_(2,3,8,9)).Interestingly, both of these compounds exhibit antimicrobial activityequal to that of compound 1, but exhibit superior selectivity. It alsoappears that antimicrobial activity is decreased with increasing achiralresidues, an effect that could be due to the reduced hydrophobicity anddecreased organizational structure of the helix. The sequence containingall Nsdp residues is relatively inactive, but that containing 4 Nsdpresidues along one face (1-Nsdp_(2,5,8,11)) exhibits antimicrobialactivity.

Several compounds were made to investigate how increasing the number ofcharges and distributing them on multiple faces effects activity. Twomolecules were increased in length in order to add hydrophobicity tocounteract the increased charges. Interestingly, no molecule in thisfamily was shown to be active against E. coli, likely due to thedecreased hydrophobicity, but all are very active against B. subtilis.At the same time, all of them are completely nontoxic to botherythrocytes and mammalian cells. It is possible that this family actsvia a different mechanism of action than less highly charged molecules.

The results of the broad spectrum antimicrobial testing and in vivoefficacy study demonstrate the therapeutic potential of antimicrobialpeptoids. Nearly all seven peptoids tested were more potent against thenine Gram positive strains (including four vancomycin-resistant) thanpexiganan. Against Gram negative strains, compounds 1 and1-Nsdp_(2,5,8,11) showed activity comparable to that of pexiganan.

The in vivo results shown in FIG. 2 demonstrate that the bacterialcounts from the peritoneal lavage were significantly reduced upontreatment with peptoid 1 (p<0.05). Additionally, no mice treated withpeptoid 1 died prematurely. Of the eleven mice treated with peptoid1-NhiS_(6,12) on the other hand, eight died prematurely during theexposure period. Of the 15 saline treated controls across all threeexperiments, only 4 died prematurely. Also, the average bacterial countwas not reduced in comparison to saline treated controls.

In summary, the results suggest that antibacterial activity among AMPsand ampetoids alike is dependent on moderate hydrophobicity and netcationic charge, while hemolytic activity is associated primarily withhigh hydrophobicity and a strongly amphipathic structure, regardless ofhelical content. MTS assays demonstrate a similar trend betweenhemolytic activity and cytotoxicity against A549 eukaryotic mammaliancells. The relationships between structure and function in ampetoids areempirically analogous to those in AMPs. X-ray reflectivity studies,which show that ampetoid 1 is membrane-active and adopts a stablemembrane-bound orientation, demonstrate molecular-level analogy betweenAMPs and ampetoids.

Results with alkylated variants demonstrate that alkyl chain attachmentmay be used to create very short peptoids that are as antimicrobiallyactive and non-toxic as longer unalkylated helices. In particular, bothNdec-1_(6mer) and Ntridec-1_(4mer) were found to be comparable inactivity and selectivity to 1. Given that the lengths of most AMPs rangefrom ˜12-100 amino acids, it is notable that such low molecular weight(<1 kDa) peptoids exhibit such potency and selectivity against bothbacteria and fungi. N-terminal attachment of alkyl tails can be a usefulmotif for improving the potency and decreasing the molecular weight ofampetoids. Moreover, alkylation is an effective and tunable modificationthat exhibits a clear effect on peptoid function.

Peptoids have greater potential than peptides to be used aspharmaceuticals and in biomaterials due to their improved stability,bioavailability, and highly tunable side chain chemistry. Sincepeptides' potential for toxicity is a major obstacle limiting theirclinical use, ampetoids' low cytotoxicity observed here relative to theAMP pexiganan further emphasizes their therapeutic potential. Theresults reported herein will aid in the rational design and optimizationof ampetoids and other non-natural oligomers as antimicrobials in thefuture.

As a continuation of the study described above, various other ampetoidswere created with the goal of more fully exploring how and to whatextent specific structural characteristics influence selectivity. (SeeTables 5-7, FIGS. 9-17 and Examples 10-15.) Additional ampetoids werederived from the previously reported dodecamer, peptoid 1, which iscomposed of ⅓ lysine-like charged monomers (Nlys) and ⅔phenylalanine-like hydrophobic monomers (Nspe) arranged in a repeatingsequence H-(Nlys-Nspe-Nspe)₄-NH (FIG. 9, Table 5). With reference tocertain compounds previously discussed, additional molecules weredesigned to explore the importance of (1) primary sequence, (2) sequenceregister, (3) net charge, and (4) charge-to-length ratio (CTLR) onantimicrobial activities and cell selectivities. The effects ofdifferent hydrophobic moieties were evaluated by systematicallyreplacing one or more Nspe monomers with other hydrophobic moieties: (1)L-proline monomers (2) achiral hydrophobic monomers, (3) oppositechirality hydrophobic monomers (Nrpe), and (4) aliphatic hydrophobicmonomers. Again, as above, Pexiganan, a widely studied andclinically-relevant AMP analog of magainin-2, was included in this studyas a basis of comparison to AMPs.

Schematic structures showing the three-fold periodic architecture ofpeptoid 1 (the basis sequence) and ampetoid variants, including thosediscussed above, are shown in FIG. 9, to clarify the design strategy andrelationships among ampetoid variants discussed herein. The threemolecular ampetoid faces discussed throughout this work are depicted asaligned monomers on the left, back, and right sides, as shown. The fullsequence of each molecule is displayed in Table 5, along with othermolecular properties including molecular weight, net charge, CTLR, andreversed-phase HPLC (RP-HPLC) solvent composition upon elution, as ameasure of molecular hydrophobicity. In general, as shown above, thenaming convention for ameptoids includes the basis compound (1, in mostcases) followed by a description of how the sequence was modifiedcompared to the basis compound. For example, compound 1B-Nlys_(4,10)describes a variant based on the sequence of 1B in which Nlys monomerswere substituted at positions 4 and 10.

The activities of all compounds against bacterial strains and mammaliancells are summarized in Table 6. The potencies of all compounds weredetermined against representative Gram-negative (E. coli ATCC 35218) andGram-positive (B. subtilis ATCC 6633) biosafety level 1 (BSL1)organisms. In addition to hemolytic activity, the conventional measureof toxicity to mammalian cells, the effect of each ampetoid on NIH 3T3mouse fibroblast cells was determined using the MTS tetrazoliumsalt-based colorimetric assay. While hemolysis measures the degree towhich erythrocytes are lysed, the MTS assay indirectly quantifies theextent to which cellular metabolic activity is inhibited.

Because the range of hemolytic activities displayed by these moleculesis significantly greater than that of inhibitory activities, hemolysisassays provide a more sensitive gauge of activity against mammaliancells. Therefore, the selectivity of each ampetoid for various bacterialstrains compared to erythrocytes is reported as the selectivity ratio(SR), which is defined as the quotient of the 10% hemolytic dose and theminimum inhibitory concentration (MIC) for each bacterial strain.

The ampetoids in this library exhibited antibacterial potencies againstGram-negative E. coli that ranged from 3.1 μM to >100 μM. Likewise,hemolytic activity ranged from HD₁₀=16 μM to HD₁₀>200 μM and metabolicinhibitory activity from ID₅₀=4.9 μM to ID₅₀>100 μM. The breadth ofactivities and selectivities evidenced by these compounds suggests thatthis library was well designed to effect a range of responses forelucidating structure-activity relationships. Compared to 1(SR_(E. coli)=3.3), 17 of the 26 ampetoid variants demonstrate improvedselectivity for E. coli (SR_(E. coli)=6.8-26). Four ampetoids(1-Npm_(2,5,8,11), 1_(11mer), 1_(ach)-Nspe₂, 1_(ach)-Nspe₁₂) exhibitedequivalent activity (6.3 μM) and superior selectivity(SR_(E. coli)=14-26) compared to pexiganan (SR_(E. coli)=11). Incomparison to their activities against E. coli, all oligomers were morepotent against B. subtilis, with MICs ranging from 0.78 to 1.6 μM.Corresponding selectivity ratios were as high as >256.

HPLC  elution Net Compound MW Sequence (% ACN)* Charge CTLR^(§)Comparators 1 1819 H-(Nlys-Nspe-Nspe)₄-NH₂ 65.1 +4 0.33 Pexiganan 2477GIGKFLKKAKKFGKAFVKILKK (SEQ ID NO: 1)-NH₂ 50.2 +9 0.41 Register & 1B1819 H-(Nspe-Nlys-Nspe)₄-NH₂ 63.4 +4 0.33 sequence 1C 1819H-(Nspe-Nspe-Nlys)₄-NH₂ 64.8 +4 0.33 variants 1scr 1819H-(Nspe)₂-(Nlys-Nspe)₃-(Nspe)₃-Nlys-NH₂ 61.1 +4 0.33 Increased1-Nlys_(5,11) 1753 H-(Nlys-Nspe-Nspe-Nlys-Nlys-Nspe)₂-NH₂ 51.2 +6 0.50charge 1B-Nlys_(4,10) 1753 H-(Nspe-Nlys-Nspe-Nlys-Nlys-Nspe)₂-NH₂ 52.7+6 0.50 variants 1B-Nlys_(4,6,10) 1720H-(Nspe-Nlys)₂-Nlys₂-(Nspe-Nlys)₂-Nlys-Nspe)-NH₂ 45.4 +7 0.581B_(15mer)- 2204 H-(Nspe-Nlys-Nspe-Nlys-Nlys-Nspe)₂-Nspe-Nlys- 55.5 +70.47 Nlys_(4,10) Nspe-NH₂ 1B_(15mer)- 2171H-(Nspe-Nlys)₂-Nlys₂-(Nspe-Nlys)₂-Nlys-Nspe₂- 50.8 +8 0.53 Nlys_(4,6,10)Nlys-Nspe-NH₂ Length 1_(10mer) 1497 H-(Nlys-Nspe-Nspe)₃-Nlys-NH₂ 60.9 +40.40 variants 1_(11mer) 1658 H-(Nlys-Nspe-Nspe)₃-Nlys-Nspe-NH₂ 63.5 +40.36 1_(13mer) 1948 H-(Nlys-Nspe-Nspe)₄-Nlys-NH₂ 62.8 +5 0.38 Proline1-Pro₃ 1755 H-Nlys-Nspe-L-Pro-(Nlys-Nspe-Nspe)₃-NH₂ 63.0 +4 0.33containing 1-Pro₆ 1755 H-Nlys-Nspe₂-Nlys-Nspe-L-Pro-(Nlys-Nspe₂)₂-NH₂62.4 +4 0.33 variants 1-Pro₉ 1755H-(Nlys-Nspe₂)₂-Nlys-Nspe-L-Pro-(Nlys-Nspe₂)-NH₂ 62.6 +4 0.331-Pro_(3,9) 1691 H-(Nlys-Nspe-L-Pro-Nlys-Nspe-Nspe)₂-NH₂ 58.1 +4 0.33Chirality 1-Nrpe_(3,6,9,12) 1819 H-(Nlys-Nspe-Nrpe)₄-NH₂ 63.5 +4 0,33variants 1-Nrpe_(2,5,8,11) 1819 H-(Nlys-Nrpe-Nspe)₄-NH₂ 66.4 +4 0.331-Nrpe_(2,3,5,6) 1819 H-(Nlys-Nrpe-Nrpe)₂-(Nlys-Nspe-Nspe)₂-NH₂ 65.0 +40.33 Achiral 1_(ach) 1701 H-(Nlys-Npm-Npm)₄-NH₂ 59.8 +4 0.33 variants1_(ach)-Nspe₂ 1721 H-(Nlys-Nspe-Npm)-(Nlys-Npm-Npm)₃-NH₂ 60.8 +4 0.331_(ach)-Nspe₁₂ 1721 H-(Nlys-Npm-Npm)₃-(Nlys-Npm-Nspe)-NH₂ 62.0 +4 0.331-Npm_(2,3,8,9) 1763 H-(Nlys-Npm-Npm-Nlys-Nspe-Nspe)₂-NH₂ 63.3 +4 0.331-Npm_(2,5,8,11) 1763 H-(Nlys-Npm-Nspe)₄-NH₂ 63.6 +4 0.33 Aliphatic1-Nsdp_(all) 1547 H-(Nlys-Nsdp-Nsdp)₄-NH₂ 63.2 +4 0.33 variants1-Nsdp_(2,3,8,9) 1683 H-(Nlys-Nsdp-Nsdp-Nlys-Nspe-Nspe)₂-NH₂ 64.7 +40.33 1-Nsdp_(2,5,8,11) 1683 H-(Nlys-Nsdp-Nspe)₄-NH₂ 63.8 +4 0.33

TABLE 5 Sequence and molecular properties of ampetoids and comparatorpeptide pexiganan. See FIG. 9 for the structures of the peptoid monomersindicated in each sequence. HPLC elution is reported as the averagepercentage of acetonitrile in the solvent mixture upon compound elutionfor three injections. A linear acetonitrile/water (0.1% trifluoroaceticacid) gradient of 5%-95% acetonitrile over 45 minutes was run on a C18column. § CTLR stands for charge-to-length ratio, which is defined asthe ratio of the total number of charged monomers to the total number ofmonomers in each sequence. Antimicrobial Mammalian Cell ActivityActivity E. coli B. subtilis HD₁₀/ MIC MIC HD₅₀ ID₅₀ Selectivity Ratios*Compound (μM) (μM) (μM) (μM) SR_(E. coli) SR_(B. subtilis) Comparative 16.3 1.6 21/ 5.1 3.3 13 Molecules 100 Pexiganan 6.3 1.6 70/ 9 11 44 >200Register & 1B 6.3 1.6 55/>100 4.9 8.7 34 sequence 1C 6.3 1.6 25/>100 5.64.0 16 variants 1scr 6.3 1.6 64/>200 8.5 10 40 Increased net1-Nlys_(5,11) 50 1.6 >100/ 85 >2 >63 charge variants >100 1B-Nlys_(4,10)50 0.78 >200/ 83 >4 >256 >200 1B- >100 1.6 >200/ >100 Inactive >125Nlys_(4,6,10) >200 1B_(15mer)- 50 0.78 >200/ 16 >4 >256 Nlys_(4,10) >2001B_(15mer)- >100 0.78 >200/ 40 Inactive 256 Nlys_(4,6,10) >200 Lengthvariants 1_(10mer) 12.5 0.78 >200/ 54 >16 >256 >200 1_(11mer) 6.3 0.78103/>200 11 16 132 1_(13mer) 3.1 0.78 21/>100 5.6 6.8 27 Proline 1-Pro₃12.5 1.6 74/>200 12 5.9 46 containing 1-Pro₆ 12.5 1.6 83/>200 18 6.6 52variants 1-Pro₉ 12.5 1.6 165/>200 24 13 103 1-Pro_(3,9) 50 1.6 >200/ 714.0 125 >200 Chirality variants 1- 6.3 1.6 16/67 3.8 2.5 10Nrpe_(3,6,9,12) 1- 6.3 1.6 22/95 5.2 3.5 14 Nrpe_(2,5,8,11)1-Nrpe_(2,3,5,6) 6.3 0.78 25/96 4.8 4.0 32 Achiral variants 1_(ach) 12.50.78 183/>200 16 15 235 1_(ach)-Nspe₂ 6.3 0.78 160/>200 11 25 2051_(ach)-Nspe₁₂ 6.3 1.6 164/>200 15 26 103 1-Npm_(2,3,8,9) 6.3 1.639/>200 15 6.2 24 1- 6.3 1.6 87/>200 6.8 14 54 Npm_(2,5,8,11) Aliphaticvariants 1-Nsdp_(all) 25 0.78 >200/ 64 >8 256 >200 1- 12.5 0.78 77/>20019 6.2 99 Nsdp_(2,3,8,9) 1- 12.5 0.78 111/>200 20 9.7 142Nsdp_(2.5.8.11) Table 6: Activity of ampetoids and pexiganan againstbacteria and mammalian cells. Presented are the minimum inhibitoryconcentrations (MICs) against E. coli (ATCC 35218) and B. subtilis (ATCC6633), 10% and 50% hemolytic doses (HD), and 50% inhibitory doses (ID)against NIH 3T3 mouse fibroblast cells. *Selectivity ratio (SR) isdefined as the ratio of the 10% hemolytic dose to the MIC for thebacterial strain of interest.

The secondary structure of ampetoids was evaluated using circulardichroism (CD) spectroscopy in 10 mM Tris buffer (pH 7.4) and the samebuffer containing small unilamellar vesicles (SUVs) comprised of eitherPOPC/cholesterol (2:1 mole ratio) or POPE/POPG (7:3 mole ratio). Whereasthe POPC/cholesterol mixture is a zwitterionic binary model lipidpreparation that mimics the membrane of erythrocytes, the negativelycharged POPE/POPG mixture mimics the composition of the E. coli outermembrane. A peptoid composed of aromatic right handedpoly-proline-type-1-like helix exhibits spectral features including amaximum at 192 nm and two local minima at ˜202 nm and ˜220 nm,respectively.

Peptoid 1, with a periodic trimer repeat sequence of Nlys-Nspe-Nspe, iscomposed of four facially-aligned Nlys monomers (positions 1, 4, 7, and10) along the left molecular face and has a charged N-terminal monomer,as shown in FIG. 9. Two isomeric variants were made in which thesequence register was modified, a change that most overtly affects therelative position of monomers with respect to the terminal positions.Peptoid 1B has a trimer repeat of Nspe-Nlys-Nspe, exhibits chargedmonomers along the back face (positions 2, 5, 8, and 11) and hashydrophobic moieties at both termini. Ampetoid 1C exhibits a sequenceregister of Nspe-Nspe-Nlys, has charged monomers along the right face(positions 3, 6, 9, and 12), and has a charged monomer at the C-terminalposition (see FIG. 9). The last variant in this family is a “scrambled”isomer, 1_(scr), which was made with a non-periodic sequence in whichthe four charged monomers are distributed across all three molecularfaces (FIG. 9).

The antimicrobial potencies against E. coli (MIC=6.3 μM) and B. subtilis(MIC=1.6 μM) of all three variants were the same as for 1, and theirtoxicities to NIH 3T3 cells were also similar, with ID₅₀ values rangingfrom 4.9-5.6 μM. The hemolytic activity, however, was reduced for1_(scr) (HD₁₀=64 μM) and 1B (HD₁₀=55 μM) compared to 1 (HD₁₀=21 μM) and1C (HD₁₀=25 μM). As a result, the selectivities for 1_(scr)(SR_(E. coli)=10) and 1B (SR_(E. coli)=8.7) were more favorable incomparison to 1 (SR_(E. coli)=3.3) and 1C (SR_(E. coli)=4.0).

CD spectroscopy showed that all these variants exhibited helicity in 10mM Tris buffer similar to that of 1, with 1_(scr) being slightly morehelical and 1C, slightly less (FIG. 10A). In both POPC/cholesterol andPOPE/POPG lipids, however, 1B exhibited significantly increased helicalintensity at 220 nm compared to the other variants (FIGS. 10B, C). Bynature of its scrambled sequence design, the extent of helicity for1_(scr) is decoupled from its degree of amphipathicity; the distributionof charges on all three molecular faces reduces its amphipathicityregardless of a three-fold periodic helical architecture. It is possiblethat the reduced intramolecular electrostatic repulsion of side chainmoieties along a given molecular face in 1_(scr) readily accommodates amore helical secondary structure, despite its overall reducedamphipathicity. Based on previous studies, reduction in amphipathicitycan improve selectivity without compromising antimicrobial activity.Indeed, 1_(scr) exhibits improved selectivity (SR_(E. coli)=10) comparedto 1 (SR_(E. coli)=3.3) with no reduction in antimicrobial activity(MIC_(E. coli)=6.3 μM for both molecules).

A second family of ampetoids was designed to evaluate the effect ofincreased charge density on potency and selectivity (FIG. 9).Structure-activity relationships derived from ampetoid variants withdecreased net charge were found to significantly reduce theirselectivity for bacteria, likely due to the less favorable electrostaticinteraction with negatively charged bacterial membranes. The variants inthis family of ampetoids were designed to explore the effect ofincreasing net charge and charge-to-length-ratio (CTLR) on cellselectivity. Ampetoid 1, and most other variants in this library, have anet charge of +4 and CTLR of 0.33. These compounds exhibit net chargesranging from +6 to +8, and CTLRs ranging from 0.47-0.58. Additionally,the hydrophobicities of these compounds (ranging from 45.4% to 55.5%acetonitrile upon HPLC elution) were all significantly reduced comparedto 1 (65.1%) (Table 9). 1-Nlys₅₋₁₁ has a total of six positive chargeswith two additional Nlys monomers (compared to 1) substituted atpositions 5 and 11, which are aligned along a back face of the helix(FIG. 9). 1B-Nlys_(4,10) is an isomeric variant of 1-Nlys₅₋₁₁ with thesequence register of 1B, and 1 B-Nlys_(4,6,10) similarly has a netcharge of +7 with cationic charge on all three molecular faces. Longer15mer variants, 1B_(15mer)—Nlys_(4,10) and 1B_(15mer)—Nlys_(4,6,10),were also made with an additional (Nspe-Nlys-Nspe) turn on theC-terminus, giving them net charges of +7 and +8, respectively (FIG. 9).

All variants in this family were significantly less active against E.coli (MIC=50->100 μM) and were non-hemolytic (HD₁₀>100 μM), a resultlargely effected by the reduced hydrophobicity of these variantscompared to 1. Because this family of variants does not exhibit a broadrange of selectivities (SR_(E. coli)>2 to >4), elucidating a meaningfulrelationship between physicochemical properties and selectivity isdifficult. A general trend observed, however, is that variants with aCTLR of less than ˜0.50, hydrophobicity greater than ˜50% acetonitrile,and one completely hydrophobic face (1-Nlys_(5,11), 1B-Nlys_(4,10), and1B_(15mer)—Nlys_(4,10)) exhibited weak activity (MIC_(E. coli)=50 μM).Variants with CTLRs greater than ˜0.50, hydrophobicity less than ˜50%acetonitrile, and had charges distributed on all three molecular faces(1B-Nlys_(4,6,10) and 1B_(15mer)—Nlys_(4,6,10)) were completely inactive(MIC_(E. coli)>100 μM). These results are commensurate with structureactivity relationships previously established for both AMPs, andampetoids: (1) antimicrobial oligomers must be sufficiently hydrophobicto be potent against Gram-negative bacteria, and (2) highly charged andpoorly amphipathic structures are generally selective.

It is most notable that the reduction in activity against E. coliexhibited by this family of molecules did not carry over to theiractivities against B. subtilis. Despite the marked change inphysicochemical properties (NC as high as +8, hydrophobicity as low as45.4% acetonitrile, and CTLR as high as 0.58) exhibited by this familyof molecules compared to 1 (NC=+4, hydrophobicity=65.1%, and CTLR=0.33),the MICs of these variants against B. subtilis were very similar to thatof 1, ranging from 0.78-1.6 μM. The resultant selectivity ratios ofmolecules in this library for B. subtilis were among the highest of allcompounds tested (SR_(B. subtilis)>256).

The CD spectra in FIG. 11 show that, with the exception of1B-Nlys_(4,6,10), all compounds exhibited helicity similar to that of 1in 10 mM Tris buffer. In POPC/cholesterol lipids, however, helicity wasinversely related to CTLR, and concomitantly, achiral monomer content.The effect of increased achiral monomer content on peptoid CD spectra isdemonstrated plainly in the hydrophobic environment of lipid vesicles;as shown in FIG. 11B, increased achiral monomer content reduced overallCD signal intensity, particularly at 220 nm.

Variants of the dodecameric peptoid 1 that ranged in length from 10-13monomers were explored as shown in FIG. 9 and Table 5. Previous studiesthat explored ampetoid length variants with a constant CTLR (1_(6mer),1_(9mer), and 1_(15mer)) showed that increased length beyond 12 monomers(ampetoid 1) only increased hemolytic activity without improvingantimicrobial potency. The variants in this study, 1_(10mer), 1_(11mer),1, and 1_(13mer) exhibit small differences in sequence length, but mostnotably effect a range of CTLRs (0.33-0.40). This range of CTLR issignificantly lower compared to the range exhibited by charge densityvariants (0.47-0.58). The CD spectra shown in FIG. 12 suggest that inboth aqueous buffer and lipid environments, all variants are similarlyhelical, and therefore exhibit similar amphipathicities.

Considering first only those variants with a net charge of +4(1_(10mer), 1_(11mer), 1), both hydrophobicity, and CTLR scalemonotonically with length. Ampetoid 1 (12mer) is the most hydrophobic(65.1% acetonitrile) and exhibits the lowest CTLR (0.33), while1_(10mer) is the least hydrophobic (60.9% acetonitrile) and ischaracterized by the highest CTLR (0.40). The data in Table 5 show thatcell selectivity is directly related to CTLR and inversely related tohydrophobicity. The slight reduction in activity of 1_(10mer) against E.coli (12.5 μM), (a result of its lowered hydrophobicity), wasaccompanied by a much larger improvement in its hemolytic activity(HD₁₀>200 μM); 1_(10mer) is therefore the most selective of this group(SR_(E. coli)>16). 1_(11mer) retained antimicrobial activity equivalentto that of 1 (MIC_(E. coli) 6.3 μM), but was less selective than1_(10mer) (SR_(E. coli)=16). Ampetoid 1 was found to be the leastselective (SR_(E. coli)=3.3). The same trend was observed forselectivity against B. subtilis (Table 6).

It is notable that based on the above correlation, 1_(13mer) (CTLR of0.38) would be expected to have an improved selectivity compared to1_(11mer) (CTLR=0.36), however this is not the case. On the contrary,ampetoid 1_(11mer) (SR_(E. coli)=16) is more selective than 1_(13mer)(SR_(E. coli)=6.8). This may be attributable to the fact that while1_(10mer), 1_(11mer), and 1 all have a net charge of +4, that of1_(13mer) is increased to +5. As discussed previously, net charge canaffect cell selectivity, particularly against Gram-negative strains.1_(11mer), with a CTLR of 0.36 and net charge of +4 represents theoptimum balance of potent antimicrobial activity and cell selectivityfrom this group of molecules. However, the selectivity ratios of1_(10mer) (SR_(E. coli)>16), 1_(11mer) (SR_(E. coli)=16), and 1_(13mer)(SR_(E. coli)=6.8) are all superior to that of 1 (SR_(E. coli)=3.3).

While proline monomers in naturally-occurring AMPs are known todestabilize α-helical secondary structure and induce the formation ofhelix-bend-helix motifs, here, proline is well accommodated in thepolyproline type-I-like peptoid helical architecture. Because ofproline's reduced hydrophobicity compared to Nspe, substitutingL-proline for a centrally-located hydrophobic residue in ampetoid 1'ssequence (variant called 1-Pro₆) was found to lower molecularhydrophobicity and improve selectivity. A family of molecules wasdesigned to evaluate how the relative position and number of prolinemonomers affects potency and selectivity, while maintaining constantCTLR and helicity. Similar to 1-Pro₆, 1-Pro₃ and 1-Pro₉ have a singleproline monomer substituted into the third and ninth positions of theampetoid 1 sequence, respectively. A fourth variant, 1-Pro_(3,9)incorporates two substituted proline monomers (FIG. 9 and Table 5). Therelative hydrophobicities of 1-Pro₃, 1-Pro₆, and 1-Pro₉ are all similar(62.4%-63%), and reduced compared to 1 (65.1%); 1-Pro_(3,9) was found tobe even less hydrophobic (58.1%). CD spectroscopy in Tris buffer andboth zwitterionic and anionic lipids show that proline-containingampetoids exhibit a similar degree of helicity as does 1 (FIG. 13).

The activity of these variants against E. coli scales withhydrophobicity; 1-Pro₃, 1-Pro₆, and 1-Pro₉ exhibited uniformly reducedpotencies against E. coli (12.5 μM) compared to peptoid 1 (6.3 μM). Thepotency of 1-Pro_(3,9) was lessened further against E. coli (50 μM).Because these variants also exhibited reduced activity against mammaliancells, selectivity was improved for all the mono-substituted variants(SR_(E. coli)=5.9-13) compared to 1 (SR_(E. coli)=3.3).

An intriguing observation regarding the mono-substitutedproline-containing variants is that the relative position of the prolinemonomer in the sequence affected cell selectivity. 1-Pro₃, 1-Pro₆,1-Pro₉ comprise a family of molecules in which CTLR, net charge, andhydrophobicity are held constant. Moreover, the degree of amphipathicityamong these variants is similar, based on the similarity of their CDspectra in both aqueous buffer and lipid environments (FIG. 13).Notably, however, shifting the proline from the N- to the C-terminalregion resulted in a progressive increase in selectivity against botherythrocytes and NIH 3T3 cells (Table 6); whereas 1-Pro₃ had an HD₁₀=74μM and ID₅₀=12 μM, those of 1-Pro₉ were 165 μM and 24 μM, respectively.The resultant selectivities monotonically increased from 1-Pro₃(SR_(E. coli)=5.9), to 1-Pro₆ (SR_(E. coli)=6.6) to 1-Pro₉(SR_(E. coli)=13). A similar trend was observed for selectivity ratiosagainst B. subtilis. This suggests that ampetoids may have a preferredorientation upon interacting with mammalian cells. If, for exampleampetoids interact with mammalian cells preferentially in the C-terminalregion, reducing hydrophobicity specifically in that portion of themolecule could impair its activity against mammalian cells and increaseselectivity.

Another strategy for improving selectivity relates to a family ofampetoids with less hydrophobic, achiral Npm side chains in place ofselected Nspe monomers in ampetoid 1 (FIG. 9 and Table 5). Becausemolecular chirality of peptoids is derived from the chirality of theside chains rather than that of the backbone, a change in the number ofchiral monomers is expected to affect the stability of the secondarystructure. This family of molecules, therefore, was designed to effect arange of decreased hydrophobicities and helicities compared to 1,independent of any change in CTLR and net charge constant. 1_(achiral)has all eight Nspe monomers replaced with Npm. Two variants,1_(ach)-Nspe₂ and 1_(ach)-Nspe₁₂ each have only one chiral Nspe monomerat the second and twelfth positions, respectively. Two other ampetoidseach contain four achiral Npm's, either aligned along the back molecularface (1-Npm_(2,5,8,11)), or distributed across both hydrophobicmolecular faces (1-Npm_(2,3,8,9)) (FIG. 9).

The hydrophobicities of these compounds ranged from 59.8% for1_(achiral) to 65.1% for peptoid 1 and generally increased with Nspecontent. Moreover, as shown in FIG. 14, the intensity of the helicalsignal, which is correlated with amphipathicity, decreased with Nspecontent. The CD spectrum of 1_(achiral) is flat, which suggests a lackof stable secondary structure and reduced molecular amphipathicity,while that of 1 exhibits the most intense spectral extrema, andcorrespondingly, the most amphipathic structure. Interestingly,1_(ach)-Nspe₁₂ showed slightly more intense CD spectra than isomericallyrelated 1_(ach)-Nspe₂, an observation that further supports a previousfinding that the C-terminal position plays a particularly important rolein stabilizing peptoid helical structure⁵⁷.

All variants with achiral side chains exhibit activities similar topeptoid 1 against E. coli (MIC=6.3-12.5 μM) and B. subtilis(MIC=0.78-1.6 μM), yet have substantially higher selectivities forbacteria over mammalian cells (SR_(E. coli)=6.2-26;SR_(B. subtilis)=24-235) (Table 6). In general, selectivity increasedwith Npm content, a monomer substitution for Nspe that simultaneouslydecreases amphipathicity and hydrophobicity.

This family of variants provides further insight into how hydrophobicityand helicity impact potency and selectivity. Previous studies have shownthat variants designed to be less hydrophobic, (via incorporation ofmore polar histidine-like side chains (e.g. 1-Nhis-6,12) or lesshydrophobic L-proline monomers (e.g. 1-Pro₆) improved selectivity, butonly at the expense of reduced antimicrobial activity. Indeed, thisobservation also held true for the proline-containing variants reportedherein. The activity and selectivity profiles of four molecules in thisfamily, however, demonstrate clearly that hydrophobicity can be reducedto improve selectivity without compromising antimicrobial activity.Variants 1_(ach)-Nspe₂, 1_(ach)-NSpe₁₂, 1-Npm_(2,5,8,11), and 1-Npm2,3,8,9) demonstrate antimicrobial activity equivalent to that of 1(MIC_(E. coli)=6.3 μM), yet significantly improved selectivities(SR_(E. coli)=6.2-26) compared to 1 (SR_(E. coli)=3.3). The most overtdifference between using achiral Npm compared to Nhis or L-Pro monomersto lower hydrophobicity is their effect on helicity. While both1-Nhis_(6,12) and 1-Pro₆ exhibited helicity equivalent to that of 1, thesubstitution of Npm monomers resulted in a progressive decrease inhelical stability, as shown in FIG. 14. This suggests that the reducingamphipathicity (correlated with helicity) concomitantly withhydrophobicity provides a means of improving selectivity withoutcompromising antimicrobial activity.

Comparison of the isomeric pairs within this group suggest that therelative position of achiral monomers does not have a significant effecton selectivity. For example, comparison of 1_(ach)-Nspe₂(SR_(E. coli)=25, ID₅₀=11 μM) and 1_(ach)-Nspe₁₂ (SR_(E. coli)=26,ID₅₀=15 μM) suggests that selectivity against both erythrocytes and NIH3T3 cells was unaffected by the position of the one chiral Nspe monomerin the sequence. Comparison of isomeric variants containing equalnumbers of Npm and Nspe monomers in different positions(1-Npm_(2,5,8,11,) and 1-Npm_(2,3,8,9)) suggest that while1-Npm_(2,5,8,11) (SR_(E. coli)=14) was more selective againsterythrocytes than 1-Npm_(2,3,8,9) (SR_(E. coli)=6.2), the opposite trendwas evident in selectivity against NIH 3T3 cells (ID₅₀=6.8 μM and 15 μM,respectively). Taken together, these results suggest that there is noclear relationship between the degree of selectivity and relativeposition of achiral monomers within the ampetoid sequence.

1_(enantiomer), a variant of peptoid 1 in which all Nspe side chainswere replaced with enantiomeric Nrpe monomers, has previously been shownto exhibit left-handed helicity, but antibacterial activities and cellselectivities congruent to those of peptoid 1. Because peptoid secondarystructure is dictated by the chirality of its side chains, it is unclearwhat the resultant secondary structure (and associatedactivity/selectivity profiles) would be in a peptoid that included bothenantiomeric side chains. Moreover, diasteriomeric peptides that containboth D- and L-amino acids were found to exhibit potent, broad-spectrumantimicrobial activity and improved selectivity. Here, variants of 1 cancontain equal numbers of Nspe and Nrpe side chains in differentarrangements as shown in FIG. 9 and Table 5. 1-Nrpe_(3,6,9,12) and1-Nrpe_(2,5,8,11) are enantiomers that have four facially-aligned Nspemonomers replaced with enantiomeric Nrpe monomers. 1-Nrpe_(2,3,5,6) hasterminally segregated enantiomeric monomers with Nrpe substitutions atpositions 2, 3, 5, and 6 in the N-terminal portion of the molecule (FIG.9).

The antibacterial potencies and cell selectivity profiles of thesevariants are very similar to that of peptoid 1, despite their disparatesecondary structures. FIG. 15 shows that in aqueous buffer as well asboth lipid environments, the enantiomeric molecules, 1-Nrpe_(3,6,9,12)and 1-Nrpe_(2,5,8,11), yield mirror image CD spectra. FIG. 15A showsthat in buffer, 1-Nrpe_(3,6,9,12) and 1-Nrpe_(2,5,8,11) appear to adopthelical secondary structures, with the overall handedness commensuratewith that of the C-terminal monomer. 1-Nrpe_(2,3,5,6,) also appears toadopt an overall right-handed spectra, but does not appear to bestrongly helical. In both zwitterionic and anionic lipids, however, theCD of the facially-aligned 1-Nrpe_(3,6,9,12) and 1-Nrpe_(2,5,8,11) weremarkedly altered, suggesting a strong interaction with lipids and asignificant change in secondary structure (FIGS. 15B and 15C).

The CD spectacle intensity for all Nrpe containing peptoids aresignificantly reduced compared to that of 1, an observation previouslycorrelated with decreased amphipathicity. However, because the CDspectra corresponding to variants in cell membrane-mimetic lipidenvironments are not typical of a peptoid helical secondary structure,it is not clear how amphipathicity is affected in these molecules.

The effect of side chain chemistry on cell selectivity, was evaluatedwith a family of ampetoids that have bulky, hydrophobic, aliphatic Nsdpside chains in place of some or all of the aromatic Nspe monomers inampetoid 1 (FIG. 9 and Table 5). The previously reported ampetoidscontaining aliphatic isoleucine-like Nssb side chains were selective,but exhibited reduced activity, particularly against Gram-negativebacterial strains. Incorporating the larger and bulkier aliphatic sidechains, 1-methylbutyl glycine (Nsmb), led to increased antibacterialpotency, but also reduced selectivity. The dipropyl glycine (Nsdp) (FIG.9) monomer used in this family of molecules is an isomer of Nsmb and wasselected to evaluate if an aliphatic monomer with branched geometrycould improve potency while maintaining favorable selectivity. Allvariants in this group had a CTLR (0.33) and net charge (+4) equivalentto that of 1.

1-Nsdp_(all) has Nsdp substituted at all eight hydrophobic monomers inthe peptoid 1 sequence. 1-Nsdp_(2,5,8,11) and 1-Nsdp_(2,3,8,9) areisomers in which half of peptoid 1's Nspe monomers were replaced withaliphatic Nsdp's. As shown in FIG. 9, 1-Nsdp_(2,5,8,11) exhibits fourNsdp monomers aligned along the back molecular face, while the fourNsdp's included in 1-Nsdp_(2,3,8,9) are distributed across bothhydrophobic faces. Whereas 1-Nsdp_(2,5,8,11) contains segregatedaromatic and aliphatic faces, 1-Nsdp_(2,3,8,9) has a mixture of aromaticand aliphatic monomers in both hydrophobic faces.

The CD spectra of right-handed helical peptoids with chiral, aliphaticside chains have been shown to be distinctly different from those witharomatic side chains in that the most pronounced spectral feature is amaximum at 210 nm. Indeed, the spectrum of 1-Nsdp_(all) exhibits thisfeature in both aqueous and lipid environments (FIG. 16).1-Nsdp_(2,5,8,11) and 1-Nsdp_(2,3,8,9,) both have spectralcharacteristics that appear to be a combination of both the aliphaticand aromatic peptoid helical signals. While the resultant “combined” CDspectra appear to be less intensely helical compared to that of 1, it isunclear how the inclusion of aliphatic and aromatic monomers affectedmolecular amphipathicity; both 1-Nsdp_(all) and 1 exhibit disparate, yethelical CD spectra.

The increase in selectivity of 1-Nsdp_(all) (SR_(E. coli)>8) wasrealized at the cost of significantly reduced activity against E. coliof (25 μM) in comparison to 1 (SR_(E. coli)=3.3; MIC_(E. coli)=6.3 μM).1-Nsdp_(all) was highly potent against B. subtilis (0.78 μM), resultingin a selectivity ratio of 256 (Table 6).

The sequences containing equal numbers of aromatic and aliphaticmonomers, 1-Nspd_(2,5,8,11) and 1-Nsdp_(2,3,8,9) exhibited slightlyreduced antimicrobial activity (MIC_(E. coli)=12.5 μM) and improvedselectivity (SR_(E. coli)=9.7 and 6.2, respectively) compared to 1(MIC_(E. coli)=6.25 μM, SR_(E. coli)=3.3). While the slightly reducedhydrophobicity could be in part responsible for the improvedselectivity, comparison with other variants suggests that the aliphaticside chain chemistry also plays an important role in its activityprofile. Unlike what has been observed in some sequences that containall aromatic hydrophobic monomers, the incorporation of aliphatic sidechains appears to improve selectivity, but only at the expense ofantimicrobial activity. For example variants 1-Npm_(2,3,8,9) (63.3%acetonitrile), 1-Npm_(2,5,8,11) (63.6% acetonitrile) and 1_(11mer)(63.5% acetonitrile) all exhibit hydrophobicities comparable to that of1-Nsdp_(2,5,8,11) (63.8% acetonitrile). The balance of the antimicrobialactivity and selectivity profiles of 1-Nsdp_(2,5,8,11), however, is lessoptimal than for sequences containing only these aromatic side chains;compared with these three molecules (MIC_(E. coli)=6.3 μM,SR_(E. coli)=6.2-16) the antimicrobial activity of 1-Nsdp_(2,5,8,11) isreduced (MIC_(E. coli)=12.5 μM) and selectivity (SR_(E. coli)=9.7)comparable. The overall hydrophobicities of these three molecules(63.2%-64.7%) were slightly reduced compared to 1 (65.1%). The reducedhydrophobicity of 1-Nsdp_(all) (63.2%) could in part be responsible forits reduced activity, however the lack of aromatic side chains may alsoplay a role. Variants containing both aliphatic and aromatic side chainsappear to provide a balance of low antimicrobial activity(MIC_(E. coli)=12.5 μM) and improved selectivity compared to 1(SR_(E. coli)=6.2-12): 1-Nsdp_(2,3,8,9) (SR_(E. coli)=6.2-12), and1-Nsdp_(2,5,8,11) (SR_(E. coli)>9.7).

The antimicrobial activity of selected ampetoids and comparator peptidepexiganan was tested against 16 clinically-relevant BSL2 bacterialstrains. The panel of bacterial strains included seven Gram-negativespecies (Proteus vulgaris, Pseudomonas aeruginosa, Proteus mirabilis,Klebsiella pneumonia, Enterobacter aerogenes, Escherichia coli, andSerratia marcescens) and nine strains from three Gram-positive species(Staphylococcus aureus, Enterococcus faecalis, and Enterococcusfaecium). Ampetoids 1,1-Pro₆, 1-Pro₉, 1_(achrial), 1-Npm_(2,5,8,11), and1-Nsdp_(2,5,8,11) were tested against these organisms. The MICs(expressed in μg/mL) are shown in Table 7A and corresponding selectivityratios (quotient of 10% hemolytic dose and MIC) are presented in Table7B.

The activities of the six peptoids tested were all similar to that thoseof pexiganan against P. vulgaris (MIC=32-64 μg/mL), K. pneumoniae(MIC=8-16 μg/mL), and E. coli (4-16 μg/mL). Against P. mirabilis and S.marcescens, pexiganan and all peptoids tested were inactive (MIC≧128).The activities of ampetoids tested against BSL2 Gram-positive strains,however, compared very favorably to those of pexiganan. The MIC ofpexiganan against the six strains of S. aureus tested ranged from 8-64μg/mL, whereas those of all the peptoids tested ranged from 4-16 μg/mL.Interestingly, 1 was uniquely active against both strains of E. faecalis(MIC=4-8 μg/mL), compared to other ampetoids (MIC=8-64 μg/mL) andpexiganan (MIC=32-128 μg/mL). All compounds were equally potent againstE. faecium (MIC=4 μg/mL).

TABLES 7A-B Broad spectrum activity and selectivity of selectedampetoids and pexiganan. (A) MICs (in μg/mL) of selected ampetoidsagainst BSL2 microbial strains. (B) Selectivity ratios are defined asthe 10% hemolytic dose divided by the MIC for the organism of interest.The hemolytic dose (μg/mL) of each compound (Table 2-2) was multipliedby its molecular weight (Table 2-1) to calculate the selectivity ratio.A Minimum inhibitory concentration (MIC) (μg/mL) Bacterial organism Pex.1 1-Pro₆ 1-Pro₉ 1_(achiral) 1-Npm_(2,5,8,11) 1-Nsdp_(2,5,8,11) P.vulgaris ATCC 32 32 32 64 32 64 32 49132 P. aeruginosa ATCC 4 8 32 32 1664 16 27853* P. mirabilis ATCC >128 >128 >128 >128 >128 >128 >128 35659K. pneumoniae 8 16 16 16 8 16 8 ATCC 33495 E. aerogenes ATCC 32 16 12864 128 64 32 35029 E. coli ATCC 25922* 8 4 16 8 16 8 8 S. marcescensATCC >128 128 >128 >128 >128 >128 >128 13880 S. aureus ATCC 29213* 32 48 16 8 8 8 VAN1^(§†) 16 4 8 8 8 8 8 VAN2^(§†) 8 4 8 8 8 8 8 NRS100(COL)^(§) 16 4 8 8 8 8 8 NRS119^(‡) 64 4 16 16 16 16 16 NRS120^(‡) 64 816 16 16 16 16 E. faecalis ATCC 29212 32 4 8 8 16 8 8 99 128 8 64 64 6464 64 E. faecium 106* 4 4 4 4 4 4 4 B Selectivity ratio (SR) Bacterialorganism Pex. 1 1-Pro₆ 1-Pro₉ 1_(achiral) 1-Npm_(2,5,8,11)1-Nsdp_(2,5,8,11) P. vulgaris ATCC 49132 5.7 1.2 4.6 5.4 9.8 2.4 5.8 P.aeruginosa ATCC 46 4.8 4.6 9.0 19 2.4 12 27853* P. mirabilis ATCC 35659<1.4 <0.3 <1.1 <2.3 <2.4 <1.2 <1.5 K. pneumoniae ATCC 23 2.4 9.1 18 399.6 23 33495 E. aerogenes ATCC 5.7 2.4 1.1 5.4 2.4 2.4 5.8 35029 E. coliATCC 25922* 23 9.5 9.1 36 19 19 23 S. marcescens ATCC <1.4 0.3 <1.1 <2.32.4 <1.2 <1.5 13880 S. aureus ATCC 29213* 5.7 9.5 18 18 39 19 23VAN1^(§†) 11 9.5 18 36 39 19 23 VAN2^(§†) 23 9.5 18 36 39 19 23 NRS100(COL)^(§) 11 9.5 18 36 39 19 23 NRS119^(‡) 2.8 9.5 9.1 18 19 9.6 12NRS120^(‡) 2.8 4.8 9.1 18 19 9.6 23 E. faecalis ATCC 29212 5.7 9.5 18 3619 19 23 99 1.4 4.8 2.3 5.4 4.9 2.4 2.9 E. faecium 106* 46 9.5 36 72 7938 46 Notes: *indicates NCCLS recommended standard strain; ^(§)indicatesmethicillian-resistant S. aureus (MRSA) strain; ^(†)indicatesvancomycin-resistant strain; ^(‡)indicates linezolid-resistant strain.

The selectivity ratios presented in Table 7B show that against mostGram-negative bacterial species, at least one ampetoid had greaterselectivity compared to pexiganan; the most favorable selectivity ratioagainst each Gram-negative species is shown in boldface type in Table7B. 1_(archiral), 1-Pro₉, and 1-Nsdp_(2,5,8,11) were the most selectiveagainst selected MDR bacterial strains. Against Gram-positive strains,ampetoids more consistently demonstrated improved selectivity comparedto pexiganan or ampetoid 1. Against all S. aureus strains, for example,the selectivity ratios of pexiganan ranged from 2.8-23, ampetoid 1ranged from 4.8-9.5, and those of the panel of more selective ampetoidsranged from 9.1-39.

As shown above, ampetoids are a new class of AMP mimics that have beenshown to exhibit potent, broad-spectrum antimicrobial activity andappear to use mechanisms of action similar to their naturalcounterparts. An understanding of factors that influence cellselectivity can be used in the context of corresponding pharmaceuticalagents. Accordingly, a library of ampetoids was designed to explore howand to what extent various physicochemical properties and structuralmotifs influenced their cell selectivity.

To broaden understanding of how ampetoids may be affecting differenttypes of mammalian cells, the hemolytic dose as well as the metabolicinhibitory dose was determined against NIH 3T3 mouse fibroblast cells.While the hemolytic and inhibitory activities exhibited similar trendsfor many ampetoids, the hemolytic dose was consistently higher than theinhibitory dose for the same compound. This is a trend that has alsobeen reported for pexiganan as well as other AMP mimics. It is possiblethat the disparity between the hemolytic and inhibitory doses is due inpart to differences in membrane composition between the two cell types.The cholesterol content of erythrocyte membranes, for example isapproximately 230 μg/mg protein, whereas NIH 3T3 cholesterol content hasbeen reported as only 30.5 μg/mg protein. The increased cholesterolcontent of erythrocytes may affect the rigidity of the membrane andoffer increased resistance to membrane-active antimicrobial agentscompared to NIH 3T3 cells.

Another disparity between hemolysis and MTS assays is that they differin terms of the measure used to quantify cytotoxicity; while hemolysismeasures the ampetoid dose needed to lyse erythrocytes, the MTS assayquantifies the dose needed to inhibit cellular metabolism, measuredindirectly by the amount of NADH produced by the cell population. It isreasonable to expect, therefore that a dose needed to lyse a cellmembrane would not necessarily be equivalent to the dose required toinhibit cellular metabolism. Whereas lysis implies membrane disruptionactivity, interference with cellular metabolism implies intracellulartargets. While animal testing would be required to determine a truetherapeutic index for these molecules, hemolysis and MTS assay resultstogether suggest that the relative effect of many ampetoids against bothcell types may be similar.

This library of ampetoids was designed to include members that exhibit avariety of structural motifs and possess physicochemical properties thatspan a wide range of values. Different ampetoids demonstrated promisingactivities against the various Gram-negative strains, but were mostpotent against K. pneumonia (ATCC 33495) and E. coli (ATCC 25922). It isparticularly notable that all 26 ampetoid variants were consistentlyvery potent against the Gram-positive screening organism, B. subtilis.The broad-spectrum testing results of selected ampetoids against MDRstrains of S. aureus, E. faecalis, and E. faecium show that ampetoidsare also potent against MRSA (4-8 μM) as well as vancomycin- andlinezolid-resistant organisms (4-8 μM and 4-16 μM, respectively). Thecorresponding selectivity ratios of 1_(achiral) (19-39) and 1-Pro₉(18-36) against MDR S. aureus strains are particularly favorablecompared to those of pexiganan (2.8-23) or ampetoid 1 (4.8-9.5). Theburden of MDR Gram-positive infections on the healthcare system issignificant and only increasing; these results suggest that ampetoidscan be a viable alternative to conventional therapies to address thisunmet clinical need.

Structure-activity studies have shown that the antibacterial activityand selectivity profiles of ampetoids are governed by thephysicochemical properties that, in a similar manner, dictate theactivity and selectivity of AMPs. The structure-activity relationshipsgleaned from these studies not only provide further evidence tore-affirm these findings in ampetoids, but also provide additionalinsight into principles that influence how more subtle changes relatedto the number, sequence position, arrangement and chemical structure ofspecific structural moieties influence activity and selectivity.

Potent, but non-selective AMPs and ampetoids tend to be hydrophobic andadopt well-defined amphipathic structures, while more selective AMPs andampetoids are typically highly cationic, exhibit only moderatehydrophobicity, and are often less amphipathic. As discussed previously,the activity and selectivity results of this study, which includedseveral new ampetoid sequences, re-affirm these general relationships inseveral ways: (1) Comparison of the less amphipathic, more selective1_(ser) to 1; (2) Comparison of the highly charged, less amphipathicvariants (1-Nlys_(5,11), 1B-Nlys_(4,10), and 1B_(15mer)—Nlys_(4,10),1B-Nys_(4,6,10) and 1B_(15mer)—Nlys_(4,6,10)) compared to 1, (3)Comparison of less hydrophobic proline-containing variants (1-Pro₃,1-Pro₆, 1-Pro₉, 1-Pro_(3,9)) to 1 (4) Comparison among achiral variants(1_(achiral), 1_(ach)-Nspe₂, 1_(ach)-Nspe₁₂, 1-Npm_(2,3,8,9),1-Npm_(2,5,8,11)), which exhibit a range of hydrophobicities andamphipathicities that scale with selectivity. Taking a closer look atmore specific structural characteristics of the ampetoids included inthis study, the influence of subtle molecular changes on activity andselectivity can be elucidated.

Comparison among the mono-substituted proline monomers suggests that theposition of monomers along the length of the molecule and can impactselectivity. The mono-substituted proline monomers exhibitedprogressively increased selectivity as the less hydrophobic prolinemonomer was moved from the N-terminal toward the C-terminal region.

Trends observed in the selectivity profiles of sequence registervariants (1, 1B and 1C) as well as length variants, 1_(13mer), suggestthat monomer position with respect to the termini also influencesselectivity. All variants are similarly potent against E. coli: theMIC_(E. coli) of 1, 1B, and 1C was 6.3 μM, while that of 1_(13mer) wasimproved by one dilution (3.1 μM). While 1 and 1C each have one chargedNlys monomer at the N and C-termini, respectively, 1B has hydrophobicNspe monomers at both termini. Conversely, 1_(13mer) has charged Nlysmonomers at both terminal positions. Consider these molecules in twogroups: (1) those with both termini charged or hydrophobic (1B and1_(13mer)), and (2) those with one charged and one hydrophobic terminus(1 and 1C). Interestingly, the hydrophobicity of those with dissimilartermini, 1 (65.1% acetonitrile) and 1C (64.8% acetonitrile) is greaterthan that of variants with like monomers at terminal positions, 1B(63.4% acetonitrile) and 1_(13mer) (62.8% acetonitrile).Correspondingly, the selectivity of the less hydrophobic variants 1B and1_(13mer) (SR_(E. coli)=6.8-8.7, SR_(B. subtilis)=27-34) is improvedcompared to that of 1 and 1C(SR_(E. coli)=3.3-4.0,SR_(B. subtilis)=13-16). This suggests that having similarly chargedtermini (either both hydrophobic or both positively charged) reducesmolecular hydrophobicity and results in an improvement in selectivity.This could be related to a similar phenomenon reported for antimicrobialpeptide analogs of magainins, which found that the relative position ofhydrophobic monomers in the sequence can impact resultant hydrophobicityand cell selectivity. These results indicate that a strategy forimproving selectivity, while maintaining antimicrobial activity, is todesign the sequence with similarly charged or similarly hydrophobicterminal monomers.

As discussed previously, the achiral family of variants provide evidencethat suggests the incorporation of achiral Npm hydrophobic monomers inplace of chiral Nspe monomers is another means of improving selectivitywithout compromising antimicrobial activity. Variants in which as manyas seven of the eight Nspe's in ampetoid 1's structure exhibitedequivalent activity and significantly improved selectivity. It appearsthat the decrease amphipathicity that occurs concomitantly withincreased Npm content results in a favorable selectivity profile. It ispossible that the less rigid structure of ampetoids with increasedachiral monomer content is less able to penetrate the rigid cellmembranes of mammalian cells.

A third way in which this library of compounds was designed to affectselectivity is through increased CTLR. The increased charge densityvariants, with CTLRs in the range of 0.47-0.58 (compared to 0.33 forampetoid 1), were at best mildly active against E. coli (MIC=50 to >100μM). Length variants, however, which were designed to effect a change inCTLR over a lower range (0.33-0.40). Of these variants, 1_(11mer) wasfound to exhibit the most optimum balance of CTLR (0.36) and sufficienthydrophobicity to permeabilize bacterial membranes (63.5%) at a lowminimum inhibitory concentrations (MIC_(E. coli)=6.3 μM).

Two other means of reducing hydrophobicity, substituting in L-prolinecontent or aliphantic Nsdp monomers, are less favorable because theyimprove selectivity at the expense of antimicrobial activity againstGram-negative bacteria. The addition of one proline monomer (1-Pro₃,1-Pro₆, and 1-Pro₉, MIC_(E. coli)=12.5 μM) and two proline monomers(1-Pro_(3,9), MIC_(E. coli)=50 μM) progressively decreased activitycompared to that of ampetoid 1 (MIC_(E. coli)=6.3 μM). Molecules withfour (1-Nsdp_(2,3,8,9) and 1-Nsdp_(2,5,8,11), MIC_(E. coli)=12.5 μM) andeight (1-Nsdp_(all), MIC_(E. coli)=25 μM) aliphatic monomers exhibited asimilar trend in reduced antimicrobial activity.

Another observation is evident from the characterization of 1_(achiral),1-Nrpe_(3,6,9,12), and 1-Nrpe_(2,5,8,11), which suggests that ampetoidscan exhibit potent antimicrobial activity without necessarily adopting astable helical secondary structure. As the name suggests, 1_(achiral) isdevoid of any chiral monomers and is thus not optically active; theresultant CD spectra in aqueous buffer and lipid environments is flat(FIG. 13). While it is conceivable that 1_(achiral) could transientlyadopt a helical structure of either handedness, there does not appear tobe external or intrinsic force to stabilize its structure.1-Nrpe_(3,6,9,12), and 1-Nrpe_(2,5,8,11), on the other hand, areenantiomeric molecules that contain equal numbers of Nspe and Nrpehydrophobic aromatic side chains. Interestingly, the overall chiralityof these monomers appears to be dictated by the chirality of the monomerin the 12^(th) position, a finding commensurate with the observationthat 1_(ach)-Nspe₁₂ exhibits a larger degree of right-handed chiralitythan does 1_(ach)-Nspe₂. This provides further evidence that theC-terminal monomer heavily influences structural stability. In buffer,both of these variants produce CD spectra that resemble that of apeptoid polyproline-type-I-like structure with the extrema normally at202 nm blue-shifted to approximately 195 nm (FIG. 15). In neutralPOPC/cholesterol lipids, however, the extrema at 220 nm is greatlydiminished, and in POPE/POPG SUVs, this feature is completelyeliminated. This marked change in CD spectra suggests that1-Nrpe_(3,6,9,12), and 1-Nrpe_(2,5,8,11) interact strongly with both ofthese lipid mixtures such that their overall structure is significantlyaltered. Taken together, it is interesting that 1_(achiral), whichappears to lack a stable secondary structure, as well as1-Nrpe_(3,6,9,12), and 1-Nrpe_(2,5,8,11), which have a CD spectra inlipids that are distinct from that of a canonical peptoidpolyproline-type-I-like, are all equally potent as ampetoid 1 against E.coli (MIC=6.3 μM). A stable helical secondary structure does not appearto be necessary for ampetoid antimicrobial activity. This finding that astable helical secondary structure does not appear to be necessary forampetoid antimicrobial activity goes beyond previous findings, whichsuggested that helicity is important only as a means of organizing anamphipathic structure.

A pair of molecules was designed to evaluate if the facial segregationaliphatic and aromatic hydrophobic monomers impacts selectivity. While1-Nsdp_(2,5,8,11) has four substituted aliphatic monomers aligned alongmolecular faces, 1-Nsdp_(2,3,8,9) has a mixture of aliphatic andaromatic hydrophobic monomers on both faces (FIG. 9). Thefacially-aligned isomer 1-Nsdp_(2,5,8,11) (SR_(E. coli)=9.7;SR_(B. subtilis)=142) was more selective than its facially-distributedcounterparts (1-Nsdp_(2,3,8,9)−SR_(E. coli)=6.2, SR_(B. subtilis)=99).While these isomers exhibit similar net charges (both +4), CTLRs (both0.33), and hydrophobicities (63.8%-64.7% acetonitrile), a notabledifference between them is that while the arrangement of hydrophobicmonomers on 1-Nsdp_(2,5,8,11), preserved one wholly aromatic face, thatof 1-Nsdp_(2,3,8,9) exhibits no completely aromatic face. It has beenshown previously that the inclusion of at least one aromatic faceincreases helical stability⁵⁷; the more intense helicity of1-Nsdp_(2,5,8,11) compared to 1-Nsdp_(2,3,8,9) in both zwitterionic andanionic lipid mixtures supports this observation (FIG. 16). Thissuggests that preservation of at least one ampetoid aromatic face mayincrease its selectivity independent of changes in other physicochemicalparameters.

The most promising therapeutic agents exhibit are highly potent againstbacteria and are nontoxic to mammalian cells. This relationship isdepicted graphically in FIG. 17, in which the hemolytic dose is plottedversus the E. coli (FIG. 17A) and B. subtilis (FIG. 17B) minimuminhibitory concentrations for selected peptoids. Peptoid 1 and pexigananare depicted by red markers for reference. Those peptoids located in thelower right coordinate space have the most promising therapeuticpotential. Many compounds reported herein exhibit more favorableactivity profiles than peptoid I and pexiganan. It is most notable thatthe increased charge density variants, which were completelynon-hemolytic, demonstrated a marked improvement in activity profileagainst Gram-positive B. subtilis.

As shown, ampetoids are a promising class of AMP mimics that exhibitpotent, broad-spectrum activity, particularly against many multi-drugresistant Gram-positive organisms. Of the 26 sequences presented here,17 demonstrate improved selectivity for E. coli compared to the basissequence, 1. The structure-activity relationships derived from thislibrary of compounds reaffirm and extend the analogy between themechanism of action of AMPs and ampetoids. Selective ampetoids tended tobe only moderately hydrophobic and amphipathic, while non-selectiveampetoids were highly hydrophobic and exhibit more highly amphipathicstructures. The relationships among ampetoid variants in this libraryalso point to the effects of how more subtle changes in sequence, sidechain chemistry, and monomer position effect selectivity. Threestrategies to improve ampetoid selectivity without compromisingselectivity include (1) Positioning of similarly cationic or hydrophobicmonomers at the sequence terminal positions, (2) Inclusion ofhydrophobic achiral Npm monomers in place of Nspe monomers (3)Optimizing the CTLR while retaining sufficient hydrophobicity topermeabilize bacterial cell membranes. Two approaches, specificallydesigned to reduce hydrophobicity effected the desired outcome ofimproving selectivity, but only at the cost of reduced antimicrobialactivity. This less optimal activity profile resulted from (1)Substitution of less hydrophobic L-proline monomers as well as (2)Substitution of aliphatic Nsdp monomers. Interestingly, while therelative position along the helix of some monomers played a role inselectivity (e.g. mono-proline substituted variants), this was notalways the case (e.g. achiral variants). The preservation of at leastone aromatic face may also play a role in increasing selectivity.Lastly, thus study provides evidence that antimicrobial activity can bemaintained in ampetoids which lack a stable secondary structure(1_(achiral)) or appear to adopt a secondary structure different fromthat of the canonical peptoid helix (1-Nrpe_(2,5,8,11) and1-Nrpe_(3,6,9,12)). Because peptoids are sequence-specific biopolymersthat can be made from a diversity of primary amines, it is conceivablethat ampetoid potency and selectivity could be finely tuned to fightspecific, clinically-relevant organisms. The design heuristicsestablished herein may aid in the design of potent, yet selective,future generations of ampetoids.

Examples of the Invention

The following non-limiting examples and data illustrate various aspectsand features relating to the compounds and/or methods of the presentinvention, including the preparation of various helical peptoidcompounds, as are available through the synthetic methodology describedherein. In comparison with the prior art, the present peptoid compoundsprovide results and data which are surprising, unexpected and contraryto the prior art. While the utility of this invention is illustratedthrough the use of several compounds, it will be understood by thoseskilled in the art that comparable results are obtainable with variousother compounds, peptoid lengths, residue sequences and/or N-pendantside chains, as are commensurate with the scope of this invention.

Example 1

Synthesis and purification. Peptoids were synthesized either using anABI 433A peptide synthesizer or a parallel synthesis robot on Rink amideresin according to the submonomer method. (See, e.g., Zuckermann, R. N.,Kerr, J. M., Kent, S. B. H., & Moos, W. H. (1992) J. Am. Chem. Soc.,114, 10646-10647.) Briefly, the amide on the nascent chain isbromoacetylated, followed by S_(N)2 displacement of bromide by a primaryamine to form the side chain. Peptides were synthesized using standardFmoc chemistry. Following synthesis, peptoids and peptides were cleavedand deprotected in trifluoroacetic acid (TFA):triisopropylsilane:water(95:2.5:2.5 by vol.) for 10 min. Compounds were purified to >97%homogeneity by RP-HPLC on a C18 column with a linear acetonitrile/water(0.1% TFA) gradient. Mass spectrometry was used to confirm the molecularweight of the purified product.

The submonomer method is illustrated graphically in FIG. 8. Each monomerof the growing peptoid polymer is assembled in two steps, using tworeadily available submonomeric units. Rink amide resin isbromoacetylated, using diisopropylcarbodiimide-activated bromoaceticacid. Next, the bromoacetylated resin undergoes S_(N)2 displacement ofbromide by a primary amine, which introduces the desired side chain.Hundreds of potential amine submonomers and corresponding side chainsare commercially or synthetically available. As a result, (1) thesynthesis of peptoids by the submonomer protocol provides facile accessto sequences of greater chemical diversity than readily obtained via themonomer approach; and (2) more directly applicable to this invention,the biomimetic peptoids are limited only by sequence order, lengthand/or N-pendant side chain structure sufficient to provide desiredantibacterial activity.

More specifically, Rink Amide resin(4-(2′,4′-Dimethoxyphenyl-(9-fluorenylmethyloxycarbonyl)-aminomethyl)-phenoxy resin, 0.25 mmol; Novabiochem) can beinitially swelled for 30 min in CH₂Cl₂. Following the resin swelling,the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is removed bytreatment with 20% piperidine solution in 1-methyl-2-pyrrolidone (NMP).The resin-bound deprotected amine is then bromoacetylated by reactionwith 4.2 ml of 1.2 M bromoacetic acid (50 mmol) in N,N-dimethylformamide(DMF) and 1.0 ml (11 mmol) neat N,N′-diisopropylcarbodiimide (DIC) for60 minutes at room temperature with constant mixing. Next, the resin isrinsed with DMF (3×10 ml), followed by NMP rinses (3×10 ml). 6 ml of a 1M solution (6 mmol) of a primary amine “submonomer” (see below) ineither NMP or CH₂Cl₂ reacted with the resin-bound bromoacetyl moiety,displacing bromide. A protected submonomer(N-tert-butoxycarbonyl-1,4-butanediamine) is synthesized in order tocreate the N-(4-aminobutyl)glycine residue (Nlys). The resin is thenrinsed again with NMP (3×10 ml) followed by DMF (3×10 ml). The productof these two reactions generates a peptoid “residue”, the identity ofwhich depended upon the submonomer amine employed. Peptoids areelongated by this submonomer method until the desired chain-length wasattained.

Following synthesis, peptoid oligomers can be cleaved from the resin,simultaneously removing the tert-butoxycarbonyl (Boc) protecting groupfrom Nlys residues, by treatment with 10 ml 2,2,2-trifluoroacetic acid(TFA)/triisopropylsilane/H₂O (95:2.5:2.5 by volume) for 30 min. Thecleavage mixture is then diluted with 25 ml 50% aqueous acetonitrile,frozen, and lyophilized. Dilution and lyophilization is repeated twicemore in order to remove excess TFA. Subsequent to cleavage, peptoids areeach purified to >97% homogeneity by preparative scale reversed-phaseHPLC using a Waters Prep LC 4000 system, with Waters 2487dual-wavelength UV detection, and gradient elution (solvent A, 0.1 vol %trifluoroacetic acid (TFA) in water; solvent B, 0.1 vol % TFA inacetonitrile) through a Vydac (Hysperia, Calif.) 214TP101550 C4peptide/protein column (10-15 μm, 300 Å, 5×25 cm). Following prep HPLC,peptoid purities and crude yields were determined from analytical scaleHPLC, performed with a Waters 2695 Separations Module with a Waters 2487dual-wavelength UV detector and gradient elution (solvent A, 0.1 vol %TFA in water; solvent B, 0.1 vol % TFA in acetonitrile) through a VydacC4 214TP53 peptide/protein column (5 μm, 300 Å, 3.2×250 mm). The precisegradient employed for HPLC depended on the identity and hydrophobicityof the oligomer in question. The composition of the HPLC solvent atgradient elution is an indication of this hydrophobicity, and isprovided in Table 1. All analytical HPLC was performed at 0.5 ml/minflow and 58° C. Preparative HPLC was performed at 50 ml/min flow androom temperature.

Such synthesis and characterization are also described in U.S. Pat. No.6,887,845, the entirety of which is incorporated herein by reference. Asillustrated therein and as would be understood by those skilled in theart made aware of this invention, the present N-substituted glycineresidues and resulting peptoid compounds are limited only by syntheticor commercial availability of the corresponding amine reagents.

Example 2

SUV preparation. Lipid mixtures, either POPE/POPG (7:3) or POPC/CH(1:1), were dissolved in chloroform, dried under N₂, and lyophilizedovernight. The resulting lipid film was hydrated with 10 mM Tris-HCl (pH7.4) at 40° C. for one hour. The resulting multilamellar vesiclesuspension was vortexed, then sonicated at 40° C. until the solutionclarified to make SUVs, which were used within 6 hours.

Example 3

CD spectroscopy. CD measurements were performed on a Jasco 715spectropolarimeter, using a quartz cylindrical cell (path length=0.02cm), with 50 μM peptoid in 10 mM Tris-HCl (pH 7.4) and 5 mM lipids whenSUVs were used. Scans were conducted at 100 nm/min between 185 and 280nm with 0.2 nm data pitch, 1 nm bandwidth, 2 s response, 100 mdegsensitivity, and 40 accumulations.

Example 4

Antibacterial assays. MICs were determined according to CLSI M7-A6protocols in a 96-well microtiter plate. In test wells, 50 μL bacterialinoculum (5×10⁵ CFU/ml) in Mueller-Hinton broth (MHB) was added to 50 μLpeptoid solution in MHB (prepared by 1:2 serial dilutions). Positivecontrols contained 50 μL inoculum and 50 L MHB without peptoid. The MICwas defined as the lowest concentration of peptoid that completelyinhibited bacterial growth after incubation at 35° C. for 16 h. MICvalues reported were reproducible between three independent experimentalreplicates, each consisting of two parallel trials. Broad-spectrumantimicrobial susceptibility testing was performed against BSL2pathogens by Nova Biologicals, Inc. (Conroe, Tex.).

Example 5

Antifungal Assays. MICs were determined using the broth microdilutionassay given by CLSI M27-A2 protocols. Candida albicans (SC5314) wasgrown on Sabouraud dextrose agar for 24 hours at 30° C. Cells weresuspended in 0.145 M saline, and the cell concentration was adjusted to3×10⁶ cells/mL. After adjusting the cell concentration, the suspensionwas diluted 1:1000 with RPMI 1640 (with L-glutamine and without sodiumbicarbonate, Invitrogen) buffered with 0.145 M 3-(N-morpholino)propanesulfonic acid (MOPS). Two-fold serial dilutions of peptoids,peptides, and amphotericin B (Calbiochem) were prepared in RPMI 1640 andmixed with an equal volume of the cell suspension in 96-well plates. Thefinal testing concentrations for the peptoids and peptides were 0.20 to100 μM, and concentrations for amphotericin B were 0.031 to 16 μM.Growth controls and sterility controls were also included. Plates wereincubated for 48 hours at 35° C., and growth of C. albicans wasinspected visually to determine the MICs. The MIC for each compound wasdefined as the lowest concentration with no visible fungal growth.Experiments were performed in duplicate on two separate days.

Example 6

Hemolysis assays. Erythrocytes were isolated from freshly drawn,heparanized human blood and resuspended to 20 vol % in PBS (pH 7.4). Ina 96-well microtiter plate, 100 L erythrocyte suspension was added to100 μL peptoid solution in PBS (prepared by 1:2 serial dilutions), or100 μL PBS in the case of negative controls. 100% hemolysis wellscontained 100 μL blood cell suspension with 100 μL 0.2 vol % TritonX-100. The plate was incubated for 1 h at 37° C., then each well wasdiluted with 150 μL PBS. The plate was then centrifuged at 1200 g for 15min, 100 μL of the supernatant from each well transferred to a freshmicrotiter plate, and A₃₅₀ measured. Percent hemolysis was determined as(A−A₀)/(A_(total)−A₀)×100, where A is the absorbance of the test well,A₀ the absorbance of the negative controls, and A_(total) the absorbanceof 100% hemolysis wells, all at 350 nm.

Example 7

MTS Assays. A549 carcinoma-derived lung epithelial cells (ATTC CCL-185)were cultured in Ham's F12K media (ATTC, Manassas, Va.). A peptoidsolution plate (100 μL/well) was prepared by serial dilution of aqueouspeptoid stocks in media. Peptoid solutions were transferred onto a96-well plate of day-old cell monolayers containing 100 μL/well mediawith ˜5000 cells/well. MTS reagent (Promega Corporation, Madison, Wis.)(40 μL/well) was added to each well and the plate was incubated at 37°C. for 3 h, after which absorbance at 490 nm was determined. Percentinhibition was determined as[1−(A−A_(test blank))/(A_(control)−A_(blank))]×100, where A is theabsorbance of the test well and A_(control) the average absorbance ofwells with cells exposed to media and MTS (no peptoid). A_(test blank)(media, MTS, and peptoid) and A_(blank) (media and MTS) were backgroundabsorbances measured in the absence of cells. The average of sixreplicates are reported, and error bars show one standard deviation.

Example 8

Specular X-ray reflectivity. XR experiments were carried out at the 9-IDbeamline at the Advanced Photon Source, Argonne National Laboratory(Argonne, Ill.). The custom-built Langmuir trough was mounted in ahelium-filled, sealed canister and equipped with a moveable singlebarrier. The surface pressure was measured using a Wilhelmy plate.Constant-pressure insertion experiments were performed at roomtemperature on Dulbecco's PBS (D-PBS) subphase. DPPG (Avanti PolarLipids, Alabaster, Al) was dissolved to a known concentration in 65/35(v/v) chloroform/methanol, then spread at the air-buffer interface usinga glass syringe; organic solvent was allowed to evaporate for 10minutes. The monolayer was compressed to the surface pressure thought tooccur in cell membranes, 30 mN/m, and XR was performed on the pure lipidlayer. Then, peptoid 1 dissolved in D-PBS was injected into the subphaseto a total concentration of 6.26 μM (well above the MICs), and allowedto insert for approximately 45 minutes, after which XR measurements wereagain collected. The X-ray reflectivity (XR) profile was determined bythe Fourier transform of the gradient of the electron densityperpendicular to the interface. XR measurements were carried out over arange of angles corresponding to q_(z) values of ˜0-0.6 Å⁻¹, whereq_(z)=(4π/λ)sin(α), λ is the wavelength, and α is the angle.

Example 9

Murine intraperitoneal infection model. Bacteria was prepared byinoculating 5 mL of MHB with a single colony of Staphyloccus aureus(ATCC #25923) from a freshly streaked plate and grown overnight at 37C.The following morning, the bacteria was subcultured by diluting ⅓ in MHBand grown for approximately 1.5 hours. The bacteria were then diluted10-fold in a 5% mucin in PBS solution and thoroughly mixed. A sample wasreserved to later determine the amount of bacteria the mice received.

Mice were weighed, marked, and injected I.P. with 200 uL of S. aureusinoculum. Four hours post infection, mice were treated I.P. with 4 mg/kgpeptoid (˜100 ug per mouse). The infection is allowed to proceedovernight. Mice were euthanized by CO₂ asphyxiation after 24 hours, andthe peritoneal cavity was exposed and lavaged with 5 mL PBS. The lavagewas mixed and reserved on ice until plating. The lavages were diluted to10⁻⁵ (in 1/10 increments) in PBS, and all dilutions were plated onto MHagar, in duplicate. 50 uL of lavage was spot plated, allowed to dry, andincubated overnight at 37° C. Colonies were counted the following day,and the CFU/ml of each sample was calculated. Plates that had too manycolonies to count were assigned an arbitrary number of 1000 colonies.

Example 10

Compound synthesis and purification. Peptoids were synthesized using anABI 433A peptide synthesizer (Applied Biosystems, Inc.) on Rink amideMBHA resin (Novabiochem, Inc.) using the submonomer approach³. Briefly,bromoacetic acid, activated by diisopropylcarbodiimide was used to forma bromocetylated intermediate on the terminal amide group. Bromide wasthen substituted with the desired primary amine through S_(N)2displacement to build the peptoid chain. The amines used in peptoidsynthesis include benzylamine, octadecylamine,(s)-(+)-2-amino-3-methylbutane, (s)-α-methylbenzylamine,(r)-(α-methylbenzylamine (all purchased from Sigma-Aldrich), andN-tert-butoxycarbonyl-1,4 diaminobutane (Nlys) that was made using apublished procedure⁸³. Resin-bound peptoids were then exposed to amixture of trifluoroacetic acid (TFA): triisopropylsilane:water(95:2.5:2.5, v:v:v) for ten minutes to cleave peptoids from the solidphase. Peptoids were purified by reversed-phase HPLC(RP-HPLC) (WatersCorporation) using a C18 column and a linear acetonitrile/watergradient. A final purity greater than 97% as measured by analyticalRP-HPLC (Waters Corporation) was achieved, and the identity of eachmolecule was checked using electrospray ionization mass spectrometry.All reagents were purchased from Sigma Aldrich.

Example 11

Circular dichroism spectroscopy. A Jasco 715 spectropolarimeter was usedto perform all CD measurements in a cylindrical quartz cell with a pathlength of 0.02 cm. Measurements were taken over the range of 190 nm to280 nm at a scanning rate of 100 nm/min. Other parameters include datapitch of 0.2 nm, bandwidth of 1 nm, response time of 2 seconds, andsensitivity of 100 mdeg. Compound concentration was 60 M in 10 mM Trisbuffer (pH 7.4). For samples in the presence of SUVs, the lipidconcentration was 5 mM. 40 accumulations were collected for each sample.

Example 12

Screening antibacterial assays. MICs were determined in 96-wellmicrotiter plates in accordance with CLSI M7-A6 protocols. Peptoidsolutions with 50 μL total volume were prepared using 2:1 serialdilutions. 50 μL of bacteria inoculum (1×10⁶ CFU/mL) prepared incation-adjusted Mueller-Hinton broth (CAMHB) was added to test wells.Control wells contained 100 μL MHB (no growth) or 50 μL inoculums with50 μL MHB with no peptoid. The MIC was taken as the lowest concentrationof peptoid that completely inhibited bacterial growth after 16 hours ofincubation at 35° C. Reported values were reproducible over threeexperiments, each containing two parallel trials.

Example 13

Broad-spectrum antibacterial assays. MICs of compounds were determinedby microdilution procedure in Mueller-Hinton broth (MHB) in accordancewith CLSI M7-A6 protocols in a manner similar to that described for thescreen antibacterial assays. Inoculated microtiter plates were incubatedat 35° C. for 24 hours prior to the result being recorded. Four ATCCstrains that were used as standards are recommended by CLSI: Pseudomonasaeruginosa ATCC 27853, Escherichia coli ATCC 25922, Staphylococcusaureus ATCC 29213 and Enterococcus faecalis ATCC 29212. Other strainsfrom the ATCC collection include Proteus vulgaris ATCC 49132, Proteusmirabilis ATCC 35659, Klebsiella pneumoniae ATCC 33495, Enterobacteraerogenes ATCC 35029 and Serratia marcescens ATCC 13880. S. aureusNRS100 (COL) is a well characterized methicillian-resistant S. aureus(MRSA) strain. The strains S. aureus VAN1 and S. aureus VAN2,vancomycin-resistant MRSA strains that were isolated in Michigan andPennsylvania, were the first vancomycin-resistant strains clinicallyisolated. S. aureus NRS119 and S. aureus NRS120 are linezolid-resistantisolates from the Network on Antimicrobial Resistance in S. aureus(NARSA) collection. E. faecalis 99 and E. faecium 106 arevancomycin-resistant enterococcal strains.

Example 14

Hemolysis assays. Erythrocytes were isolated from freshly drawn,heparanized human blood and resuspended in PBS (pH 7.4) to make a 20%volume suspension. Peptoid solutions were prepared by serial dilution(2:1) in a 96-well microtiter plate. For test wells, 100 μL oferythrocyte suspension was added to 100 μL of peptoid solution in PBS;PBS without peptoid was used as the negative control and 0.2 vol %Triton X-100 as the positive control that indicates 100% hemolysis.After 1 hour incubation at 37° C., each well was diluted with 150 μLPBS. Plates were then centrifuged at 1,200×g for 15 minutes to pelletthe cells. 30 μL of the supernatant from each well were transferred tothe corresponding well of a second 96-well plate that contains 70 μLPBS. Using a plate reader, the absorbance at 350 nm was measured, andpercent hemolysis was defined as (A−A₀)/(A_(total)−A₀)×100, where A isthe absorbance of the test well, A₀ the average absorbance of negativecontrols, and A_(total) the average absorbance of 100% hemolysis wells.

Example 15

MTS assays. NIH/3T3 cells (ATCC Corporation) cultured at 37° C. and 5%CO₂ in complete Dulbecco's modified eagle's media (CDMEM) supplementedwith 1% sodium pyruvate, 1% penicillin-streptomycin, 1.5 g/L NaHCO₃, and10% fetal bovine serum. Cells were seeded at a density of 5,000 cellsper well for NIH/3T3 cells in 96-well plates (100 μl total volume). Apeptoid solution plate (100 μL total volume per well) was prepared byserial dilution of aqueous peptoid stock solution in Hank's balancedsalt solution (HBSS) media. The day-old cell monolayers were washed withHBSS and media was replaced with 100 μL HBSS. The contents of thepeptoid solution plate were transferred onto corresponding wells of thecell monolayer plate, and 40 μL MTS reagent (Promega, Inc.) was added toeach well. After incubating for 3 hours at 37° C., absorbance at 490 nmwas determined. The percentage inhibition was determined as[1−(A−A_(testblank))/(A_(control)−A_(blank))]×100, where A is theabsorbance of the test well and A_(control) the average absorbance ofthe wells with cells exposed to media and MTS (no peptoid).A_(testblank) (media, MTS, and peptoid) and A_(blank) (media and MTS)were measured as background absorbances in the absence of cells. Theaverage of six replicates is reported.

We claim:
 1. A poly-N-substituted glycine antibiotic compound of aformula

wherein A is selected from H and a terminal N-alkyl substituted glycineresidue, where said alkyl substituent is selected from about C₄ to aboutC₂₀ linear, branched and cyclic alkyl moieties; n is an integer selectedfrom 1-3; B is selected from NH₂, one and two N-substituted glycineresidues, said N-substituents independently selected from naturalα-amino acid side chain moieties, isomers and carbon homologs thereof;X, Y and Z are independently selected from N-substituted glycineresidues, said N-substituents independently selected from naturalα-amino acid side chain moieties, isomers and carbon homologs thereof,and proline residues, at least one of said X, Y and Z residues isN_(Lys) and at least one said N-substituent is chiral, said compound ofa formula